Receptor targeting constructs and uses thereof

ABSTRACT

Disclosed herein are drug delivery molecules that comprise a ligand that targets a cell surface molecule; a membrane penetration domain; and a payload binding domain; and pharmaceutical compositions comprising the same. Also disclosed are methods of treating cancer, inhibiting the progression of cancer, preventing cancer metastasis, and delivering a therapeutic compound to the brain in a subject in need thereof, the methods comprising identifying a subject in need thereof; providing a composition comprising the drug delivery molecule as disclosed herein; and administering an effective amount of the composition to the subject.

RELATED APPLICATIONS

The present invention is filed under 35 U.S.C. § 371 as the U.S.national phase of International Application No. PCT/US2015/011870, filedJan. 16, 2015, which designated the U.S. and claims priority from theU.S. Provisional Application Ser. No. 61/928,903, filed on Jan. 17,2014, by Lali K. MEDINA-KAUWE et al. and entitled “c-MET TARGETINGCONSTRUCT AND USES THEREOF,” the entire disclosure of which isincorporated herein by reference, including the drawings.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No. CA140995and Grant No. CA129822 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 15, 2016, isnamed EOS_005_US_SeqListing.txt and is 29 kilobytes in size.

FIELD OF THE INVENTION

The invention relates to the field of biotechnology. Specifically, theinvention relates to compositions that deliver therapeutic agents totarget cells, such as cancer cells.

BACKGROUND OF THE DISCLOSURE

Many tumors that resist or acquire resistance to current targetedtherapies used in the clinic exhibit elevated surface levels of proteinssuch as c-Met. For example, lung cancer acquires resistance to EGF-Rinhibitors such as Tarceva. Inhibitors like Tarceva are intended toblock the activity of receptor tyrosine kinases (known as tyrosinekinase inhibitors, or TKI's), but the majority of cases do not respondto TK inhibition. These tumors are characterized by elevated levels ofcell surface proteins (such as c-Met) and thus become excellentcandidates for therapeutic approaches described herein which approachescan target the overexpressed proteins and penetrate tumor cells.

Current attempts are being made in the field to develop c-Met antibodiesor inhibitors that are intended to block signaling through c-Met.However, past history indicates that the majority of cases will notrespond to signal blocking antibodies or small molecules because thetumor adopts alternative means to continue proliferating despite signalinhibition.

The compositions described herein circumvent the need to block signalingby using a cell surface receptor (for example, c-Met) as a portal todeliver toxic molecules into the tumor cell and kill tumors from within.Ligand directed delivery enables the targeted binding to tumors that arepositive for specific cell surface receptors (for example, c-Met) andthe membrane penetration domain in the delivery molecule enablespenetration and lysis across the endosomal membrane after cell surfacereceptor-mediated endocytosis. The delivery protein is also modified tonon-covalently assemble with and transport certain therapeutic moleculesthrough, for example, ionic interactions.

SUMMARY OF THE INVENTION

Disclosed herein are drug delivery molecules that comprise a ligand thattargets a cell surface molecule; a membrane penetration domain; and apayload binding domain; and pharmaceutical compositions comprising thesame. Also disclosed are methods of treating cancer, inhibiting theprogression of cancer, preventing cancer metastasis, and delivering atherapeutic compound to the brain in a subject in need thereof, themethods comprising identifying a subject in need thereof; providing acomposition comprising the drug delivery molecule as disclosed herein;and administering an effective amount of the composition to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the uptake of a single-strandedoligonucleotide using Lipofectamine as control (FIG. 1A) and HerPBK10(FIG. 1B).

FIG. 2 shows that PBK10 can deliver a synthetic mRNA encoding GFP. FIG.2A provides a summary of mRNA used in this experiment. FIG. 2B shows GFPexpression from mRNA delivered by Lipofectin (right) in comparison aGFP-expressing plasmid delivered by Lipofectin (left). FIG. 2C is aschematic of PBK10-mRNA complexes. FIG. 2D shows the cell binding datafor PBK10-mRNA complexes. FIG. 2E shows the results of the uptake ofcell-bound complexes. FIG. 2F shows the image of the results of theexpression of GFP, whereas FIG. 2G depicts the same data in a graphform.

FIG. 3 is a schematic of (A) InlB321, which encompasses the minimaldomain for c-MET receptor binding; and (B) InlB321 bound to theextracellular domain of c-MET.

FIG. 4 is a schematic showing a recombinant gene construct is assembledto encode a new fusion protein, InlB-PBK10. A. Construction ofpRSET-InlB-PBK10. B. Construction of pRSET-GFP-InlB. C. Confirmation ofcloning inserts by restriction digest.

FIG. 5 is the results of a Western blot showing the recombinant proteinsInlB, InlB-PBK10 and GFP-InlB are produced in bacteria.

FIG. 6 is a graph showing the surface level of c-MET varies amongdifferent tumor and non-tumor cell lines. Relative receptor levels onthe surface of non-permeabilized cells as measured by a cell surfaceELISA performed in a 96-well format are shown. ELISA results show thatthe H1993 (lung cancer cell line) and MDA-MB-231 (breast cancer cellline) are among the cells with the highest cell surface levels of c-MET.RANKL (prostate cancer cell line) and MDA-MB-435 (breast cancer cellline) display moderate levels while LN-GFP (prostate cancer cell line)and Cos-7 (African green monkey kidney fibroblast) display low levels ofcell surface c-MET.

FIG. 7 shows the results of the experiments showing the InlB-derivedpeptide recognizes c-MET. A. InlB321 peptide shows preferential bindingto c-MET positive, but not low c-MET, cells. Fluorescence activated cellsorting (FACS) was used to measure the relative level of InlB(recognized by immunofluorescence) bound to c-MET positive cells. TheFACS data shows a relatively higher level of InlB binding to high c-METcells (H1993) compared to low c-MET cells (LN GFP). B. Confirmation ofc-MET binding by competitive inhibition. InlB binding to H1993 could beinhibited when free InlB was pre-incubated with a soluble peptidederived from the extracellular, ligand binding domain of c-MET (MET)before binding to the cells. InlB and MET were incubated at a 1:1 molarratio (MET:InlB), which predicts a 50% reduction in receptor binding ifInlB is specific to MET. C. Cell binding by InlB-PBK10 is proportionalto c-MET levels. InlB-PBK10 showed a higher level of binding to thecells with higher c-MET cell surface expression (MDA-MB-231) incomparison to cells expressing relatively low c-MET levels (Cos-7) asmeasured by cell surface ELISA. D. Inhibition of binding to c-MET+ cellsby competing ligand. Escalating concentrations of free InlB ligand waspre-bound to MDA-MB-231 cells for 1 h on ice before addition ofInlB-PBK10 when increasing concentrations of InlB were pre-bound to thecells. E. InlB-PBK10 undergoes receptor-specific binding to c-MET+ cellsin suspension. MDA-MB-435 cells in suspension were incubated withincreasing concentrations of free InlB ligand and after removing unboundInlB, cells were incubated with InlB-PBK10. The concentrations of freeInlB ligand were chosen so that the molar ratios of InlB-PBK10 to InlBwere: 1:1, 1:5, and 1:10. Western blotting was performed to measure therelative InlB-PBK10 levels co-precipitating with the cell pellets.Densitometric measurements (right panel) of immunoblot bands show thatlevels of InlB-PBK10 binding decreased as the concentration of InlBincreased, consistent with InlB-PBK10 binding to c-MET.

FIG. 8 shows that InlB-PBK10 internalizes into c-MET+ cells.

FIG. 9 shows InlB-PBK10 can deliver toxic molecules to c-MET+ cells. A.Preparation of InlB-PBK10-Ga particles. Schematic shows procedure ofisolating particles by ultrafiltration after mixing InlB-PBK10 withGa-corrole to promote non-covalent assembly. B. DLS of InlB-PBK10-Gaparticles. C. InlB-PBK10 mediates cytosolic entry of the corrolepayload. D. I-Dox reduces survival of c-MET positive tumor cells. E.Free InlB Inhibits I-Dox toxicity.

FIG. 10 shows a Xenogen Spectum image for the biodistribution ofInlB-PBK10 after systemic (tail vein) delivery in a nu/nu mouse bearingsubcutaneous bilateral flank MDA-MB-435 tumors. A. Images of the wholemouse at indicated time points after tail vein injection. Blue arrowspoint to kidneys. White arrows point to tumors. B. Images of the tumorsand tissues harvested from the same mouse sacrificed after the 4 h timepoint.

FIG. 11 depicts the assembly of HerMn. A. Schematic of HerPBK10 protein,highlighting functional domains. B. Chemical structure of Mn-corrole(S2Mn). C Schematic of non-covalent assembly. D. TEM (inset) and dynamiclight scattering (DSL) measurement of HerMn particles in solution.

FIG. 12 is a group of graphs showing HerPBK10 binds to HER3 and is notinhibited by patient serum. A. ELISA of HerPBK10 binding to immobilizedHER3 (human ErbB3 extracellular domain; Prospec) −/+pre-incubation withsoluble HER3 peptide as a competitive inhibitor (HER3 block). Un: noHerPBK10. B. ELISA of HerPBK10 binding to HER2+ cells −/+pre-incubationwith: a 1× and 10× molar ratio of soluble HER3 peptide, soluble HER4peptide (ERBB4 peptide, Abnova), betacellulin (10 μg/mL), or pertuzumab(Pz) as competitive inhibitors. C. ELISA of HerPBK10 binding to HER2+(MDA-MB-435) cells in serum from five HER2+ patients and age matchedcontrols (HER2−). Control samples were bound in bovine serum andreceptor-binding verified by competitive inhibition with recombinantheregulin ligand (+Her). N=3. *, p<0.05 compared to control (−Her: nocompetitive inhibitor).

FIG. 13 shows the results that HerPBK10 binds to mouse HER3. A. Aminoacid sequence alignment of domains I-II (aa 20-239, heregulin-bindingdomain) of human and mouse HER3. Blue residues indicate amino aciddifferences. B. Relative HER3 levels detected by ELISA (withoutpermeabilization) using an anti-HER3 antibody that cross-reacts withboth human and mouse HER3 (1B2E; Cell Signaling Technologies). C.Binding of HerPBK10 to 4T1 mouse mammary tumor cells. N=3. *, p<0.05compared to HerPBK10 alone

FIG. 14 is a graph showing the data for HerMn toxicity on human HER2+and HER2− tumor cells.

FIG. 15 are photographs showing the mechanism of HerMn cytotoxicity. A.Confocal fluorescence images showing reduction of mitochondrial membranepotential by HerMn in MDA-MB-435 cells. B. Confocal fluorescence imagesshowing superoxide-mediated collapse of actin (red) and tubulin (green)by HerMn.

FIG. 16 shows the data that S2Ga interacts with TSPO. A-B. Retentateswere evaluated for the presence of TSPO-bound corrole by measuring theabsorbance and fluorescence spectra. C-D. Evidence of HerGa interactionwith TSPO in situ. The green fluorescent JC-1 dye used in C fluorescesred when accumulated into mitochondria. D is a quantification of redfluorescence in C. *, p<0.05.

FIG. 17 shows the biodistribution in tumor-bearing mice. Xenogen imagingand quantification of Alexa680-labeled HerMn, trastuzumab (Tz) and BSA(12 nmol ea) after tail vein injection. Graph shows meanfluorescence−/+SEM.

FIG. 18 shows the data for the therapeutic efficacy of HerMn. A. HER2+MDA-MB-435 tumor growth in female nude mice receiving daily IV (via tailvein) injections of HerMn or S2Mn (5 nmoles corrole/injection) once/dayfor 6 consecutive days. Control groups received saline or HerPBK10 atequivalent conc to HerMn. Treatments began at ˜200 mm3 ave tumor vol.Tumor volumes were measured before (day 1), during (day 3), and after(days 8, 15, and 22) injections of reagents. N=8-10 tumors/group.*p<0.05 (one-way ANOVA). B. Human CDC viability during exposure toHerMn, S2Mn, HerPBK10, or doxorubicin (Dox) for 2 days (solid lines) or5 days (dashed lines). N=3 per conc, from three separate experiments

FIG. 19 shows the tissue distribution of HerPBK10 in mice with notumors.

DETAILED DESCRIPTION OF THE EMBODIMENTS Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

“Beneficial results” may include, but are in no way limited to,lessening or alleviating the severity of the disease condition,preventing the disease condition from worsening, curing the diseasecondition, preventing the disease condition from developing, loweringthe chances of a patient developing the disease condition and prolonginga patient's life or life expectancy. In some embodiments, the diseasecondition is cancer. In some embodiments, the disease condition is anautoimmune disease.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to leukemia,myeloma, B-cell lymphomas (Hodgkin's lymphomas and/or non-Hodgkinslymphomas), brain cancer, breast cancer, colorectal cancer, lung cancer,hepatocellular cancer, kidney cancer, gastric cancer, pancreatic cancer,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma,head and neck cancer, brain cancer, prostate cancer, androgen-dependentprostate cancer, and androgen-independent prostate cancer.

“Chemotherapeutic drugs” or “chemotherapeutic agents” as used hereinrefer to drugs used to treat cancer including but not limited toAlbumin-bound paclitaxel (nab-paclitaxel), Actinomycin, Alitretinoin,All-trans retinoic acid, Azacitidine, Azathioprine, Bevacizumab,Bexatotene, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cetuximab,Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin,Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone,Erlotinib, Etoposide, Fluorouracil, Gefitinib, Gemcitabine, Hydroxyurea,Idarubicin, Imatinib, Ipilimumab, Irinotecan, Mechlorethamine,Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab,Ofatumumab, Oxaliplatin, Paclitaxel, Panitumab, Pemetrexed, Rituximab,Tafluposide, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin,Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine,Vorinostat, Romidepsin, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP),Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin,Mitomycin, ixabepilone, Estramustine, or a combination thereof.

“Subject” or “individual” or “animal” or “patient” or “mammal,” is meantany subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. In some embodiments, the subject hascancer. In some embodiments, the subject had cancer at some point in thesubject's lifetime. In various embodiments, the subject's cancer is inremission, is re-current or is non-recurrent.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans, domestic animals, farm animals,zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs,rabbits, rats, mice, horses, cattle, cows; primates such as apes,monkeys, orangutans, and chimpanzees; canids such as dogs and wolves;felids such as cats, lions, and tigers; equids such as horses, donkeys,and zebras; food animals such as cows, pigs, and sheep; ungulates suchas deer and giraffes; rodents such as mice, rats, hamsters and guineapigs; and so on. In certain embodiments, the mammal is a human subject.The term does not denote a particular age or sex. Thus, adult andnewborn subjects, as well as fetuses, whether male or female, areintended to be included within the scope of this term.

“Treatment,” “treating,” “therapy,” or “therapeutic,” as used hereinrefer to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition, prevent the pathologic condition, pursueor obtain beneficial results, or lower the chances of the individualdeveloping the condition even if the treatment is ultimatelyunsuccessful. Those in need of treatment include those already with thecondition as well as those prone to have the condition or those in whomthe condition is to be prevented. A particular procedure oradministration is considered therapeutic even if, following theadministration, the subject does not feel better. Thus, any ameliorationof the subject's disease state, or any slowing of the progression of thedisease, is considered therapeutic. Examples of cancer treatmentinclude, but are not limited to, active surveillance, observation,surgical intervention, chemotherapy, immunotherapy, radiation therapy(such as external beam radiation, stereotactic radiosurgery (gammaknife), and fractionated stereotactic radiotherapy (FSR)), focaltherapy, systemic therapy, vaccine therapies, viral therapies, moleculartargeted therapies, or a combination thereof.

“Tumor,” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

“Therapeutic agents” as used herein refers to agents that are used to,for example, treat, inhibit, prevent, mitigate the effects of, reducethe severity of, reduce the likelihood of developing, slow theprogression of and/or cure, a disease. Diseases targeted by thetherapeutic agents include but are not limited to carcinomas, sarcomas,lymphomas, leukemia, germ cell tumors, blastomas, antigens expressed onvarious immune cells, and antigens expressed on cells associated withvarious hematologic diseases, autoimmune diseases, and/or inflammatorydiseases.

By “about” a value in the context of the present disclosure it is meantthat the disclosure encompasses the listed value ±25%, or alternativelythe listed value ±15%, or alternatively the listed value ±10%, oralternatively the listed value ±5%. Thus, for example, by “about 50amino acids” it is meant 50±25% amino acids (i.e., a range of 37-63amino acids), or alternatively 50±15% amino acids (i.e., a range of42-58 amino acids), or alternatively 50±10% amino acids (i.e., a rangeof 45-55 amino acids), or alternatively 50±5% amino acids (i.e., a rangeof 47-53 amino acids).

Drug Delivery Molecules

Described herein are drug delivery molecules. The drug delivery moleculeincludes a ligand that targets a cell surface molecule, a membranepenetration domain and a payload binding domain. The ligand in the drugdelivery molecule delivers the molecule to the target cell, such as acancer cell. The membrane penetration domain mediates cytosolicpenetration of the target cell. The payload binding domain forms acomplex with a therapeutic agent. The drug delivery molecule in complexwith the therapeutic molecule delivers the therapeutic agent to thetarget cell, such as a cancer cell. In various embodiments, the cancercell is any one or more of leukemia, myeloma, B-cell lymphomas(Hodgkin's lymphomas and/or non-Hodgkins lymphomas), brain cancer,breast cancer, colorectal cancer, lung cancer, hepatocellular cancer,kidney cancer, gastric cancer, pancreatic cancer, cervical cancer,ovarian cancer, liver cancer, bladder cancer, cancer of the urinarytract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neckcancer, brain cancer, prostate cancer, androgen-dependent prostatecancer, and androgen-independent prostate cancer.

In some embodiments, the drug delivery molecules disclosed herewith formnanoparticles having between about 5 nm to about 50 nm in diameter. Insome embodiments, the nanoparticles have a size of between about 10 toabout 30 nm. The payload, i.e., the therapeutic agent, is then bound tothe nanoparticle. In one embodiment, the nanoparticle comprises ametallated corrole, for example manganese (Mn), iron (Fe), or gallium(Ga) corrole. In other embodiments, the nanoparticle comprises a proteinor a protein fragment. In some embodiments, the binding of thetherapeutic agent to the nanoparticle is through a process selected fromthe group consisting of electrostatic interactions, hydrophobicinteractions, hydrophilic interactions, hydrogen bonding, and covalentbonding. Once the nanoparticle-therapeutic agent combination enters acell, the bond between the nanoparticle and the therapeutic agent isbroken, either due to the conditions within the cell that disrupt theassociation of the nanoparticle and the therapeutic agent, or becausethe covalent bond between the two is hydrolyzed by an enzyme.

In some embodiments, the cell surface molecule is a receptor that isimplicated in a signal transduction pathway that leads to reduced oreliminated apoptosis. Examples of cell surface molecules that may betargeted by a ligand in the drug delivery molecule described hereininclude, but are not limited to, any one or more of 4-1BB, 5T4,adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242antigen, CA-125, carbonic anhydrase 9 (CA-IX), c-MET, CCR4, CD152, CD19,CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8),CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888,CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folatereceptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu,hepatocyte growth factor (HGF), human scatter factor receptor kinase,IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growthfactor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1,mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R α, PDL192,phosphatidylserine, prostatic carcinoma cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1,TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, vimentin orcombinations thereof. Other target molecules or particles specific forcancer will be apparent to those of skill in the art and may be used inconnection with alternate embodiments of the invention. In anembodiment, the ligand targets c-MET on cancer cells. In an embodiment,the ligand is HGF. In an embodiment, the ligand is Internalin B or afragment thereof or a variant thereof. In another embodiment, the ligandis the bacterial invasin (Inv) protein.

Thus, in some embodiments, the ligand is a natural binding partner, or amolecule that can bind to the cell surface molecule. In someembodiments, the ligand is a protein, protein fragment, polypeptide, oroligopeptide. In other embodiments, the ligand is an antibody or anantibody fragment. In certain other embodiments, the ligand is a smallorganic molecule that binds to the cell surface molecule. In someembodiments, the small organic molecule mimics the structure of anatural binding partner for the target cell surface molecule and bindscompetitively to the target cell surface molecule, while in otherembodiments, the small organic molecule binds non-competitively to thetarget cell surface molecule.

In some embodiments, the membrane penetration domain is a protein,protein fragment, polypeptide, or oligopeptide. In certain embodiments,the membrane penetration domain is a polypeptide having between about 3to about 35 amino acids.

In an embodiment, the membrane penetration domain is the penton baseprotein or a fragment thereof from Adenovirus. The penton base proteinnormally mediates cell-binding, entry and cytosolic penetration ofadenovirus (for example, adenovirus serotype 5) during the early stagesof infection. The penton base may comprise an RGD motif (Arg-Gly-Asp).As used herein, “PB” refers to a penton base segment.

In some embodiments, the payload binding domain is a protein, proteinfragment, polypeptide, or oligopeptide. In certain embodiments, themembrane penetration domain is a polypeptide having between about 3 toabout 35 amino acids.

In one embodiment, the payload binding domain is a decalysine motif,also referred to as “K10.” The decalysine motif comprises ten lysineresidues.

In some embodiments, the payload that binds to the payload bindingdomain binds is a nucleic acid. In some embodiments, the nucleic acid isselected from the group consisting of double stranded deoxyribonucleicacid (dsDNA), single stranded deoxyribonucleic acid (ssDNA), ribonucleicacid (RNA), messenger ribonucleic acid (mRNA), transfer ribonucleic acid(tRNA), ribosomal ribonucleic acid (rRNA), small interfering ribonucleicacid (siRNA), single stranded ribonucleic acid (ssRNA), andoligonucleotides (whether single stranded or double stranded).

In certain embodiments, the binding of the payload binding domain to thepayload is through a process selected from the group consisting ofelectrostatic interactions, electrophilic interactions, hydrophilicinteractions (van der Waals forces), hydrogen binding, or covalentbinding.

In various embodiments, the ligand in the drug delivery molecule targetsa cell surface molecule on a cancer cell. In an embodiment, the cellsurface molecule is a receptor on a cancer cell.

In an embodiment, described herein is a drug delivery molecule thatincludes a ligand that targets a cell surface molecule, a membranepenetration domain and a payload binding domain, wherein the ligand isInternalin B (InlB) or a fragment thereof or a variant thereof, themembrane penetration domain is the penton base protein or a fragmentthereof and the payload binding domain is a decalysine motif. InternalinB targets the cell surface protein c-Met. The natural ligand of c-Met ishepatocyte growth factor (HGF). HGF forms a tetramer and requiresdisulfide bond formation. Internalin B, obtained from Listeriamonocytogenes also recognizes and bind c-MET, but does not tertamerizeor require disulfide bond formation. Internalin B can be expressed as afusion protein and the fusion protein also binds to c-Met. InlB does notcompete with HGF. In some embodiments, the drug delivery moleculecomprising InlB, penton base protein and decalysine motif may furthercomprise a cytotoxic agent, such as corrole compounds. Corrole compoundsare porphyrin-like molecules. These compounds can chelate a number ofdifferent metals (such as iron, gallium, manganese etc.), non-covalentlybind carrier proteins, are cytotoxic and cannot penetrate cells withoutcarrier proteins.

In other embodiments, the ligand targets the cell surface molecule CD4,or alternatively, CD19 or CD20. In further embodiments, the ligandtargets one of the human epidermal growth factor receptors (HER), forexample HER2 or HER3. In another embodiment, the ligand targets anintegrin.

In various embodiments, the drug delivery molecule further comprises atherapeutic agent. The therapeutic agent forms a complex with thepayload binding domain. In various embodiments, therapeutic agentsinclude but are not limited to alkylating agents, antimetabolites,anti-tumor antibiotics, mitotic inhibitors, corticosteroids, cytotoxicagents or combinations thereof. The complex between the therapeuticagent and the payload binding domain may be covalent or non-covalent. Insome embodiments, non-covalent complexes may be via any one or more ofvan der Waals forces, hydrogen bonding, electrostatic interactions,hydrophobic/hydrophilic interactions. In some embodiments, theinteractions between the therapeutic agent and the payload bindingdomain may be mediated by a linker. In some embodiments, the therapeuticagent is doxorubicin or a corrole compound.

Also described herein are methods for treating cancer in a subject inneed thereof. The methods comprise identifying a subject in need of thetreatment and providing a composition that includes a drug deliverymolecule and administering an effective amount of the composition to thesubject so as to treat cancer. In various embodiments, the drug deliverymolecule includes a ligand that targets a receptor on a cell surface, amembrane penetration domain and a payload binding domain and furthercomprises a therapeutic agent as described herein.

Also described herein are methods for inhibiting the progression ofcancer in a subject in need thereof. The methods comprise identifying asubject in need of the treatment and providing a composition thatincludes a drug delivery molecule and administering an effective amountof the composition to the subject so as to inhibit the progression ofcancer. In various embodiments, the drug delivery molecule includes aligand that targets a receptor on a cell surface, a membrane penetrationdomain and a payload binding domain and further comprises a therapeuticagent as described herein.

Also described herein are methods for preventing cancer metastasis in asubject in need thereof. The methods comprise identifying a subject inneed of the prevention and providing a composition that includes a drugdelivery molecule and administering an effective amount of thecomposition to the subject so as to prevent cancer metastasis. Invarious embodiments, the drug delivery molecule includes a ligand thattargets a receptor on a cell surface, a membrane penetration domain anda payload binding domain and further comprises a therapeutic agent asdescribed herein.

Also provided herein are methods for treating, inhibiting or preventingmetastasis of drug resistant cancers (for example, cancer resistant toEGFR tyrosine kinase inhibitor). The methods comprise identifying asubject in need of the treatment and providing a composition thatincludes a drug delivery molecule and administering an effective amountof the composition to the subject so as to treat, inhibit or preventmetastasis of drug resistant cancers. In various embodiments, the drugdelivery molecule includes a ligand that targets a receptor on a cellsurface, a membrane penetration domain and a payload binding domain andfurther comprises a therapeutic agent as described herein. In anembodiment, the drug resistant cancers over-express a receptor selectedfrom the group consisting of c-Met, HER2, CD4, and CD20.

Also provided herein are methods of delivering a therapeutic compound tothe brain. The methods comprise identifying a subject in need of suchdelivery and providing a composition that includes a drug deliverymolecule and administering an effective amount of the composition to thesubject. In various embodiments, the drug delivery molecule includes aligand that targets a receptor on a cell surface, a membrane penetrationdomain and a payload binding domain and further comprises a therapeuticagent as described herein. The compositions described hereinsurprisingly cross the blood-brain barrier and deliver the payload tothe cells in the brain. Thus, these compositions are uniquely suited forthe treatment of cancers, for example metastatic cancers, in the brain.

In various aspects of the therapeutic methods described herein, the drugdelivery molecule in the composition includes a ligand that targets areceptor on a cell surface, a membrane penetration domain and a payloadbinding domain, wherein the ligand is Internalin B (InlB) or a fragmentthereof or a variant thereof, the membrane penetration domain is thepenton base protein or a fragment thereof and the payload binding domainis a decalysine motif. The drug delivery molecule further comprises atherapeutic agent such as doxorubicin or corrole compounds.

Therapies

Another aspect of the invention relates to treating cancer (for example,leukemia, myeloma, B-cell lymphomas (Hodgkin's lymphomas and/ornon-Hodgkins lymphomas), brain cancer, breast cancer, colorectal cancer,lung cancer, hepatocellular cancer, kidney cancer, gastric cancer,pancreatic cancer, cervical cancer, ovarian cancer, liver cancer,bladder cancer, cancer of the urinary tract, thyroid cancer, renalcancer, carcinoma, melanoma, head and neck cancer, brain cancer,prostate cancer, androgen-dependent prostate cancer, andandrogen-independent prostate cancer) by administering an effectiveamount of a composition that includes the drug delivery moleculescomplexed with therapeutic agents as described herein. In someembodiments, the therapeutic agent can be any chemotherapeutic drug thatis applicable to treating the particular type of cancers. Thetherapeutic agent can be an organic molecule, a biological molecule(e.g., a peptide or a nucleic acid), or a combination thereof. Invarious embodiments, therapeutic agents include but are not limited toalkylating agents, antimetabolites, anti-tumor antibiotics, mitoticinhibitors, corticosteroids, cytotoxic agents or combinations thereof.In an embodiment, the therapeutic agent is a corrole compound. In anembodiment, the therapeutic agent is an siRNA molecule.

In some embodiments, the composition comprising an effective amount ofthe drug delivery molecule complexed with a therapeutic agent isadministered with one or more chemotherapeutic agents, such as those setforth herein. Effective amounts of the composition and thechemotherapeutic agent may be administered sequentially or concurrently.

In some embodiments, the administering is systemic. In some embodiments,the administering is local. A variety of means for administering thecomposition to subjects are known to those of skill in the art. In someaspects of all the embodiments of the invention, the compositions areadministered through routes, including ocular, oral, parenteral,intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol),pulmonary, cutaneous, topical, or injection administration.

Additional therapies that may be used with the compositions comprisingan effective amount of the drug delivery molecule complexed with atherapeutic agent to treat cancer include but are not limited tosurgery, radiation, immunotherapy, vaccine or combinations thereof. Theadditional therapies may be administered sequentially or simultaneouslywith therapies comprising administering an effective amounts of acompositions comprising an effective amount of the drug deliverymolecule complexed with a therapeutic agent to treat cancer (forexample, melanoma or ovarian carcinoma).

In some embodiments, chemotherapeutic agents may be selected from anyone or more of cytotoxic antibiotics, antimetabolities, anti-mitoticagents, alkylating agents, arsenic compounds, DNA topoisomeraseinhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins;and synthetic derivatives thereof. Exemplary compounds include, but arenot limited to, alkylating agents: treosulfan, and trofosfamide; plantalkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomeraseinhibitors: doxorubicin, epirubicin, etoposide, camptothecin, topotecan,irinotecan, teniposide, crisnatol, and mitomycin; anti-folates:methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs:5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs:mercaptopurine and thioguanine; DNA antimetabolites:2′-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole;and antimitotic agents: halichondrin, colchicine, and rhizoxin.Compositions comprising one or more chemotherapeutic agents (e.g., FLAG,CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside(Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine,doxorubicin, and prednisone. In another embodiments, PARP (e.g., PARP-1and/or PARP-2) inhibitors are used and such inhibitors are well known inthe art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene ResearchLaboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34(Soriano et al., 2001; Pacher et al., 2002b); 3-aminobenzamide(Trevigen); 4-amino-1,8-naphthalimide; (Trevigen);6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); andNU1025 (Bowman et al.).

As described herein, in various embodiments, therapies include, forexample, radiation therapy. The radiation used in radiation therapy canbe ionizing radiation. Radiation therapy can also be gamma rays, X-rays,or proton beams. Examples of radiation therapy include, but are notlimited to, external-beam radiation therapy, interstitial implantationof radioisotopes (I-125, palladium, iridium), radioisotopes such asstrontium-89, thoracic radiation therapy, intraperitoneal P-32 radiationtherapy, and/or total abdominal and pelvic radiation therapy. For ageneral overview of radiation therapy, see Hellman, Chapter 16:Principles of Cancer Management: Radiation Therapy, 6th edition, 2001,DeVita et al., eds., J. B. Lippencott Company, Philadelphia. Theradiation therapy can be administered as external beam radiation orteletherapy wherein the radiation is directed from a remote source. Theradiation treatment can also be administered as internal therapy orbrachytherapy wherein a radioactive source is placed inside the bodyclose to cancer cells or a tumor mass. Also encompassed is the use ofphotodynamic therapy comprising the administration of photosensitizers,such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA),phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and2BA-2-DMHA.

As described herein, in various embodiments, therapies include, forexample, immunotherapy. Immunotherapy may comprise, for example, use ofcancer vaccines and/or sensitized antigen presenting cells. Theimmunotherapy can involve passive immunity for short-term protection ofa host, achieved by the administration of pre-formed antibody directedagainst a cancer antigen or disease antigen (e.g., administration of amonoclonal antibody, optionally linked to a chemotherapeutic agent ortoxin, to a tumor antigen). Immunotherapy can also focus on using thecytotoxic lymphocyte-recognized epitopes of cancer cell lines.

As described herein, in various embodiments, therapies include, forexample, hormonal therapy, Hormonal therapeutic treatments can comprise,for example, hormonal agonists, hormonal antagonists (e.g., flutamide,bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RHantagonists), inhibitors of hormone biosynthesis and processing, andsteroids (e.g., dexamethasone, retinoids, deltoids, betamethasone,cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids,mineralocorticoids, estrogen, testosterone, progestins), vitamin Aderivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs;antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g.,cyproterone acetate).

The duration and/or dose of treatment with anti-cancer therapies mayvary according to the particular anti-cancer agent or combinationthereof. An appropriate treatment time for a particular cancertherapeutic agent will be appreciated by the skilled artisan. Theinvention contemplates the continued assessment of optimal treatmentschedules for each cancer therapeutic agent, where the genetic signatureof the cancer of the subject as determined by the methods of theinvention is a factor in determining optimal treatment doses andschedules.

In various embodiments, the subject for whom predicted efficacy of ananti-cancer therapy is determined, is a mammal (e.g., mouse, rat,primate, non-human mammal, domestic animal such as dog, cat, cow,horse), and is preferably a human. In another embodiment of the methodsof the invention, the subject has not undergone chemotherapy orradiation therapy. In alternative embodiments, the subject has undergonechemotherapy or radiation therapy (e.g., such as with cisplatin,carboplatin, and/or taxane). In related embodiments, the subject has notbeen exposed to levels of radiation or chemotoxic agents above thoseencountered generally or on average by the subjects of a species. Incertain embodiments, the subject has had surgery to remove cancerous orprecancerous tissue. In other embodiments, the cancerous tissue has notbeen removed, e.g., the cancerous tissue may be located in an inoperableregion of the body, such as in a tissue that is essential for life, orin a region where a surgical procedure would cause considerable risk ofharm to the patient, or e.g., the subject is given the anti-cancertherapy prior to removal of the cancerous tissue.

Pharmaceutical Compositions

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of the drug delivery moleculedescribed herein so as to treat cancer (for example, leukemia, myeloma,B-cell lymphomas (Hodgkin's lymphomas and/or non-Hodgkins lymphomas),brain cancer, breast cancer, colorectal cancer, lung cancer,hepatocellular cancer, kidney cancer, gastric cancer, pancreatic cancer,cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer ofthe urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma,head and neck cancer, brain cancer, prostate cancer, androgen-dependentprostate cancer, and androgen-independent prostate cancer). In variousembodiments, the drug delivery molecule includes a ligand that targets areceptor on a cell surface, a membrane penetration domain and a payloadbinding domain. The drug delivery molecule further comprises atherapeutic agent that complexes with the drug delivery molecule asdescribed herein. In various embodiments, the drug delivery moleculedelivers the therapeutic agent to the target cancer cell.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited toaerosol, nasal, oral, transmucosal, transdermal, parenteral or enteral.“Parenteral” refers to a route of administration that is generallyassociated with injection, including intraorbital, intraocular,infusion, intraarterial, intracapsular, intracardiac, intradermal,intramuscular, intraperitoneal, intrapulmonary, intraspinal,intrasternal, intrathecal, intrauterine, intravenous, subarachnoid,subcapsular, subcutaneous, transmucosal, or transtracheal. Via theparenteral route, the compositions may be in the form of solutions orsuspensions for infusion or for injection, or as lyophilized powders.Via the parenteral route, the compositions may be in the form ofsolutions or suspensions for infusion or for injection. Via the enteralroute, the pharmaceutical compositions can be in the form of tablets,gel capsules, sugar-coated tablets, syrups, suspensions, solutions,powders, granules, emulsions, microspheres or nanospheres or lipidvesicles or polymer vesicles allowing controlled release. Typically, thecompositions are administered by injection, either intravenously orintraperitoneally. Methods for these administrations are known to oneskilled in the art.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventionaltechniques of pharmacy involving milling, mixing, granulation, andcompressing, when necessary, for tablet forms; or milling, mixing andfilling for hard gelatin capsule forms. When a liquid carrier is used,the preparation will be in the form of a syrup, elixir, emulsion or anaqueous or non-aqueous suspension. Such a liquid formulation may beadministered directly p.o. or filled into a soft gelatin capsule.

The pharmaceutical compositions according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Before administration to patients, formulants may be added to theagents. A liquid formulation may be preferred. For example, theseformulants may include oils, polymers, vitamins, carbohydrates, aminoacids, salts, buffers, albumin, surfactants, bulking agents orcombinations thereof.

Carbohydrate formulants include sugar or sugar alcohols such asmonosaccharides, disaccharides, or polysaccharides, or water solubleglucans. The saccharides or glucans can include fructose, dextrose,lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran,pullulan, dextrin, alpha and beta cyclodextrin, soluble starch,hydroxethyl starch and carboxymethylcellulose, or mixtures thereof.“Sugar alcohol” is defined as a C4 to C8 hydrocarbon having an —OH groupand includes galactitol, inositol, mannitol, xylitol, sorbitol,glycerol, and arabitol. These sugars or sugar alcohols mentioned abovemay be used individually or in combination. There is no fixed limit toamount used as long as the sugar or sugar alcohol is soluble in theaqueous preparation. In one embodiment, the sugar or sugar alcoholconcentration is between 1.0 w/v % and 7.0 w/v %, more preferablebetween 2.0 and 6.0 w/v %.

Amino acids formulants include levorotary (L) forms of carnitine,arginine, and betaine; however, other amino acids may be added.

In some embodiments, polymers as formulants include polyvinylpyrrolidone(PVP) with an average molecular weight between 2,000 and 3,000, orpolyethylene glycol (PEG) with an average molecular weight between 3,000and 5,000.

It is also preferred to use a buffer in the composition to minimize pHchanges in the solution before lyophilization or after reconstitution.Most any physiological buffer may be used including but not limited tocitrate, phosphate, succinate, and glutamate buffers or mixturesthereof. In some embodiments, the concentration is from 0.01 to 0.3molar. Surfactants that can be added to the formulation are shown in EPNos. 270,799 and 268,110.

Additionally, the compositions can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Preferred polymers, and methods to attach them to peptides, areshown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546which are all hereby incorporated by reference in their entireties.Preferred polymers are polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and in someembodiments, has an average molecular weight between 1000 and 40,000,between 2000 and 20,000, or between 3,000 and 12,000. In someembodiments, PEG has at least one hydroxy group, such as a terminalhydroxy group. The hydroxy group may be activated to react with a freeamino group on the inhibitor. However, it will be understood that thetype and amount of the reactive groups may be varied to achieve acovalently conjugated PEG/antibody of the present invention.

Water soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), etc. POG is preferred. Onereason is because the glycerol backbone of polyoxyethylated glycerol isthe same backbone occurring naturally in, for example, animals andhumans in mono-, di-, triglycerides. Therefore, this branching would notnecessarily be seen as a foreign agent in the body. The POG has amolecular weight in the same range as PEG. The structure for POG isshown in Knauf et al., 1988, J. Bio. Chem. 263:15064-15070 and adiscussion of POG/IL C 2 conjugates is found in U.S. Pat. No. 4,766,106,both of which are hereby incorporated by reference in their entireties.

After the liquid pharmaceutical composition is prepared, it may belyophilized to prevent degradation and to preserve sterility. Methodsfor lyophilizing liquid compositions are known to those of ordinaryskill in the art. Just prior to use, the composition may bereconstituted with a sterile diluent (Ringer's solution, distilledwater, or sterile saline, for example) which may include additionalingredients. Upon reconstitution, the composition is administered tosubjects using those methods that are known to those skilled in the art.

The dosage and mode of administration will depend on the individual.Generally, the compositions are administered so that antibodies aregiven at a dose between 1 μg/kg and 20 mg/kg, between 20 μg/kg and 10mg/kg, between 1 mg/kg and 7 mg/kg. In some embodiments, it is given asa bolus dose, to increase circulating levels by 10-20 fold and for 4-6hours after the bolus dose. Continuous infusion may also be used afterthe bolus dose. If so, the antibodies may be infused at a dose between 5μg/kg/minute and 20 μg/kg/minute, or between 7 μg/kg/minute and 15μg/kg/minute.

Kits

The invention also provides a kit to treat, inhibit and/or preventmetastasis of cancer in a subject in need thereof. The kit comprises acomposition comprising a drug delivery molecule complexed with atherapeutic agent, as described herein and instructions for use of thecomposition for treating, inhibiting and/or preventing metastasis ofcancer in subjects in need thereof.

The kit is an assemblage of materials or components, including at leastone of the inventive compositions. Thus, in some embodiments the kitcontains a composition including a drug delivery molecule complexed witha therapeutic agent, as described above.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. In one embodiment, the kit isconfigured particularly for human subjects. In further embodiments, thekit is configured for veterinary applications, treating subjects suchas, but not limited to, farm animals, domestic animals, and laboratoryanimals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat, reduce the severity of, inhibit or prevent neutropeniain a subject. Optionally, the kit also contains other useful components,such as, measuring tools, diluents, buffers, pharmaceutically acceptablecarriers, syringes or other useful paraphernalia as will be readilyrecognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.As used herein, the term “package” refers to a suitable solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding the individual kit components. Thus, for example, a package canbe a bottle used to contain suitable quantities of an inventivecomposition containing the catalytically active antibody havingsialidase activity produced by the methods described herein. Thepackaging material generally has an external label which indicates thecontents and/or purpose of the kit and/or its components.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1: Delivery of Single-Stranded Oligonucleotide and SyntheticmRNA

The delivery of synthetic mRNA is an improved alternative to genedelivery. Gene delivery vectors must breach the nucleus to enable geneexpression, whereas mRNA only requires delivery into the cytoplasm fortranslation of protein products. Such as approach is used to express,for example, the so-called Yamanaka factors for inducing pluripotency insomatic cells. While traditional “lipofection” may be useful fordelivery of mRNA in vitro, this and similar systems are not effective invivo.

The disclosed targeted cell penetration protein, HerPBK10, has provenefficacy for nucleic acid and drug delivery in vivo. The relatedprotein, PBK10, has also been developed for gene and drug delivery.HerPBK10 facilitates targeted binding and penetration of cells viainteraction with the human epidermal growth factor receptor (HER3), andPBK10 does so via integrin interaction.

Below, the utility of HerPBK10 and PBK10 for the delivery ofsingle-stranded oligonucleotide and synthetic mRNA is demonstrated.

HerPBK10 Transports a Labeled Oligonucleotide in MDA-MB-435 Cells.

To determine whether HerPBK10 can mediate single-strandedoligonucleotide delivery, a Cy3-labeled oligonucleotide (50 pmol) wasincubated with HerPBK10 (5 μg) or Lipofectamine 2000 (Invitrogen,Carlsbad, Calif., USA), a commercial transfection reagent as acomparative control, in 0.1 M HEPES/Optimem I (Invitrogen; Carlsbad,Calif., USA) for 20 minutes at RT. The resulting mixture was added toMDA-MB-435 cells, which express HER, and incubated for 1 h at 37° C.Cells were fixed in 4% PFA for 15′ at RT and processed forimmunofluorescence against HerPBK10. Cells were counterstained with DAPIto identify nuclei. Images were acquired using a Leica SP2 laserscanning confocal microscope. Lipofectamine-mediated uptake resulted inoligonucleotide localization inside the cells, as expected (FIG. 1A).Importantly, HerPBK10 colocalized with the oligonucleotide duringbinding and uptake of HerPBK10-oligo complexes (FIG. 1B), suggestingthat HerPBK10 mediates transport of the oligonucleotide into cells.

PBK10 Mediates mRNA Delivery.

A 1 kb synthetic mRNA encoding GFP (FIG. 2A) was used to test theability of PBK10 to mediate delivery into cells for protein expression.GFP expression from mRNA delivered by Lipofectin was compared to aGFP-expressing plasmid delivered by Lipofectin. When delivered byLipofectin, the mRNA expressed GFP at levels in HeLa cells that weredetectable by fluorescence microscopy (FIG. 2B). PBK10 and syntheticmRNA encoding GFP were mixed at a 20:1 weight ratio of PBK10:mRNA for˜20 min at RT in HEPES-buffered saline (HBS) and then added to adherentHeLa cells at ˜50-70% confluency. Thus, PBK10 formed complexes with mRNAsimilar to the gene delivery complexes that PBK10 had previously beendeveloped to deliver (FIG. 2C).

The ability of PBK10 to mediate delivery was examined by first assessingthe cell binding of complexes made between PBK10 and the mRNA.PBK10-mRNA complexes were incubated on the HeLa cells on ice to promotereceptor binding but not internalization, then the cells were washed toremove free (unbound) complexes and processed by ELISA to identify cellsurface-bound complexes using a primary antibody against PBK10. AnELISA-based detection of cell surface binding showed that the complexesbind (PBK10+mRNA) to the cells (FIG. 2D). Cells were also processed forimmunofluorescence against PBK10 alone after incubation of PBK10(without mRNA) on cells. To verify uptake of cell-bound complexes (E),cells were incubated with complexes on ice as described earlier, washed,then warmed at 37° C. to promote internalization. Cells were fixed atthe indicated time points after warming and processed forimmunofluorescence against PBK10 (green). Cells were counterstained withrhodamine phalloidin and DAPI to identify actin (red) and nuclei (blue),respectively. Images were acquired using a Leica SPE laser scanningconfocal microscope. The findings show that the complexes underwenttime-dependent internalization into HeLa cells (FIG. 2E). A separate setof HeLa cells were fixed and processed for immunofluorescence againstGFP at ˜24 h after the cells were incubated with PBK10-mRNA complexes.The findings show that GFP expression was detectable afterPBK10-mediated delivery of mRNA, albeit at low frequency (few cells)(FIG. 2D). The optimal weight ratio of PBK10:mRNA to enable sufficientGFP expression (detectable by immunofluorescence) was 20 (FIG. 2E).These findings altogether show that it was possible to deliver a mRNA byPBK10 or HerPBK10, since both proteins interact with nucleic acids insimilar fashion.

Example 2: A Protein Nano-Construct Targeted to c-Met

The receptor tyrosine kinase (RTK), c-MET, and its endogenous ligand,hepatocyte growth factor (HGF), contribute to cell migration,morphogenic differentiation, and organization of three-dimensionaltubular structures as well as cell growth and angiogenesis during normaltissue development. However, dysregulation of c-MET and HGF cancontribute to tumor progression, and in such circumstances, correlateswith poor prognosis in a broad range of human cancers.

Cell surface elevation of c-MET has been associated withdrug-resistance, including acquired resistance to currentsignal-blocking therapies, and thus has become an important biomarkerfor RTK-targeted therapy. Whereas the majority of therapies targetingRTKs are designed to inhibit downstream signaling pathways that supporttumor survival, tumors that initially respond to such treatment almostuniversally acquire resistance to signal inhibition while a significantpopulation are already inherently resistant to such signal-blockingtherapies.

Tumor-targeting strategies that do not require signal inhibition mayprove more effective on c-MET positive cancer cells. This may beaddressed by ligands that recognize c-MET to trigger cell uptake ofattached therapeutics, thus bypassing the need to block signaling. WhileHGF has the potential to accomplish this, its requirement fortetramerization and disulfide bonding presents technical complicationsfor therapeutics development. An alternative, and potentially superiorligand for c-MET targeting may possibly be derived from a bacterium thatcauses food-poisoning.

The human pathogen, Listeria monocytogenes, binds c-MET to invade hostcells through its surface proteins called Internalins. Specifically,Internalin B (InlB) triggers receptor-mediated endocytosis after c-Metbinding. InlB and HGF recognize different regions of c-Met, and InlBdoes not require tetramerization or disulfide bonds for binding.Consequently, InlB may lend itself to the nanobiologic strategy fortumor-targeting that we have previously established for targeting otherreceptors such as the human epidermal growth factor receptor (HER).

PBK10 is a recombinant protein derived from the adenovirus capsid pentonbase that can mediate gene and drug delivery into cells through themembrane penetrating activity of the penton base. It has been shown thatPBK10 can be targeted to tumor cells when fused to tumor-specificligands. In this study, the receptor-binding site of InlB is produced asa recombinant fusion to PBK10 to produce the new protein, InlB-PBK10,which mediates the targeted-delivery of cytotoxic agents to c-METpositive cancer cells.

The findings show that InlB-PBK10 can be produced as a soluble fusionprotein that recognizes c-MET on a variety of tumor cell lines, andundergoes rapid internalization after cell binding. InlB-PBK10 forms˜10-20 nm diameter nanoclusters with toxic compounds such as corroles,and mediates corrole penetration into the cytoplasm after cell entry,causing tumor cell death. Thus, Inl-PBK10 is a novel construct formediating the targeted delivery of toxic molecules to MET-expressingtumors.

Background

C-MET as a Tumor Biomarker.

Mesenchymal Epithelial Transition factor or MET is a receptor tyrosinekinase (RTK) that was first discovered as an activated oncogene. Theendogenous ligand for MET, Hepatocyte Growth Factor (HGF), also known asfibroblast-derived cell motility factor or Scatter Factor (SF), normallyactivates MET to induce cell proliferation, motility, survival anddifferentiation pathways. MET and HGF are mainly expressed in cells ofepithelial and mesenchymal origin, respectively. The paracrine signalingbetween HGF and MET mediates epithelial-mesenchymal interactions thatregulate tissue growth and morphogenic differentiation. HGF-METsignaling in normal tissue contributes to embryogenesis, organogenesis,angiogenesis, wound healing and tissue regeneration, whereas aberrantsignaling of this pathway is associated with tumor development andprogression, tumor cell invasion and metastasis.

C-MET consists of an amino (N)-terminal extracellular domain, a membranespanning segment and a carboxy (C)-terminal intracellular kinase domain.The extracellular region consists of an amino (N)-terminal Semaphorin(Sema) domain adjacent to a PSI domain (present in plexins, semaphorins,and integrins) followed by four immunoglobulin (Ig)-like domains, whichtogether comprise the binding site for HGF. Receptor binding by HGFleads to dimerization of MET, activating signaling through ERK1/2, AKTand STAT3, phosphoinositide 3-kinase (PI-3K), Ras-Raf-MAPK, andphospholipase C. Ligand-triggered endocytosis of MET occurs throughdynamin and clathrin-dependent pathways that mediate trafficking throughboth degradative and recycling endocytotic routes.

Dysregulation of c-MET signaling occurs through several mechanisms,including overexpression and constitutive kinase activation with orwithout gene amplification, kinase-domain mutation, andparacrine/autocrine activation of c-MET by overexpression of HGF.Ligand-independent activation of c-MET also disrupts normal HGF-METsignaling and can result from mutations causing constitutivedimerization, as well as hypoxic conditions. The latter can activateHIF-1α-induced transcription of MET causing elevated protein levelsamplifying HGF signaling and promoting invasion.

It has been generally accepted that the heterogeneous expression ofvarious RTKs across the tumor is a major mechanism of resistance in manytypes of cancer. Therapies aimed at inhibiting one specific RTK areoften not successful due to upregulation or ligand stimulation of otherRTKs that, in turn, sustain signaling of factors critical forcell-survival, including PI3K and mitogen-activated protein kinase(MAPK). This mechanism of resistance was identified in studies showingthat resistance to EGFR inhibitors is associated with compensatoryupregulation of MET signaling in non-small cell lung cancer as well asother tumors, including breast cancer. Taken altogether, c-MET is avalid candidate for therapeutic intervention since it is associated withvarious types of cancer, poor prognosis and metastasis, and could be auseful biomarker for identifying and targeting resistant tumors.

The therapies currently targeted at the HGF/MET pathway in the clinicconsist of either antibodies directed against HGF (Ficlatuzumab) or MET(Onartuzumab), or small molecule inhibitors (Tivantinib or ARQ197)directed at the MET kinase domain. A problem with all of theseapproaches is their reliance on signal inhibition for therapeuticefficacy, which is prone to the compensatory RTK cross-talk mentionedearlier, facilitating the development of resistance. There is anincreasing interest in using combination therapies to block or disruptthe function of multiple RTKs, particularly EGF-R and MET given thesignificant cross-talk between these two receptors. While in vitrostudies testing this approach have shown promise on tumor cell linessuch as lung, breast, gastric and colorectal cancer, clinical trialstesting this approach are still underway and remain inconclusive.

A Bacterial Pathogen Protein as a Potential c-MET Targeting Agent.

Internalin B (InlB) is a protein displayed on the surface of the humanbacterial pathogen, L. monocytogenes (Lm), and facilitates the entry ofLm into non-phagocytic cells through binding to c-MET. InlB is a memberof the larger family of Lm internalin proteins which are comprised of anN-terminal helical cap domain followed by differing numbers ofleucine-rich repeats (LRRs) adjacent to an immunoglobulin-likeinter-repeat (IR) region. Unlike other internalins, InlB structure alsocontains a B-repeat and three GW modules at the C-terminal end. InlB321,a fragment of InlB capable of binding to c-MET with high affinity,consists of protein domains known as the Cap, LRR (protein-proteininteraction domain), and IR regions. The binding of InlB321 to METoccurs through its two domains: LRR and IR, which bind to the Ig1 andSema domain of MET respectively.

InlB has several advantages over HGF as a potential tumor-targetingligand. HGF is a heterodimeric protein containing 20 sulfide bonds andrequires cleavage of a pro-protein into assembled subunits, which maypose complications for recombinant production, especially as a fusion toexogenous protein domains. In contrast, InlB321 has already beenproduced as a recombinant soluble ligand in Escherichia coli and cantolerate fusion to other proteins (FIG. 3A). In vitro studies havedemonstrated that the InlB321 peptide can trigger receptor-mediatedinternalization after MET binding, which is conducive to its use as anano-delivery agent. It is also important to note that there is nooverlap between the HGF binding site of MET with that of InlB321 (FIG.3B); therefore, there is no competition between these two ligands forbinding to MET.

In the present study this peptide is re-engineered by producing it as arecombinant fusion to the Ad5 penton base (PB) that is modified by adecalysine sequence (K10) at the carboxyl (C)-terminus end. Theresulting multi-domain protein, InlB-PBK10, contains functions fortransport of anionic cargoes such nucleic acids or corroles (mediated bythe K10 domain), targeted binding and internalization (mediated by theInlB321 domain), and membrane penetration and intracellular trafficking(mediated by the PB domain). Results are presented for the constructionof the InB-PBK10 fusion gene, the production of the fusion proteinencoded by this recombinant gene, the characterization of the proteinwith respect to its ability to bind to MET and enter cells, and theevaluation of its ability to deliver cytotoxic payloads in vitro.

Results

A Recombinant Gene can be Constructed to Encode InlB-PBK10.

The strategy for producing the InlB-PBK10 gene construct involved atwo-step cloning method entailing polymerase chain reaction (PCR)amplification of InlB321 for cloning into the pRSET-A bacterialexpression plasmid, followed by insertion of PBK10 at the 3′ end of theInlB321 sequence. To accomplish this, oligonucleotide primers weredesigned to introduce restriction sites by PCR into InlB321 to join thissequence with PBK10 for “in-frame” insertion into the expressionplasmid, pRSET-A.

Our forward and reverse oligonucleotide primers contained the followingsequences respectively:

(SEQ ID NO: 1) 5′-AGTGAGCTCGAGACTATCACTGTG-3′ and (SEQ ID NO: 2)5′-GTTGGTGACTTTCTCCCACCTTCACCACCTTCATCTAGATATCCATG GTAT-3′.

The restriction site, SacI, was introduced in the forward primer toplace the InlB321 reading frame contiguous with the N-terminalpoly-histidine sequence encoded by pRSET-A. BglII and KpnI restrictionsites were introduced into the reverse primer. SacI and KpnI were usedto insert InlB321 into pRSETA, and BglII was used for subsequentinsertion of PBK10 just 3′ to the InlB321 coding sequence. A sequenceencoding a Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO:3) amino acid motif wasincluded in the reverse primer to incorporate a short flexible linkerbetween InlB321 and PBK10. A high-fidelity polymerase was used to ensurethat mutations were not introduced into the InlB321 PCR product.

The 800 bp InlB321 PCR product was ligated into pRSETA at the SacI andKpnI restriction sites, producing the plasmid construct, pRSETA-InlB.PBK10 was then inserted into pRSETA-InlB at restriction sites BglII andHindIII. To do so, PBK10 was excised from the plasmid construct,pRSETA-PBK10 at restriction sites BamHI and HindIII and ligated intodouble digested pRSETA-InlB, resulting in the pRSETA-InlB-PBK10construct (FIG. 4A). We also produced the construct, pRSETA-GFP-InlB,that encodes InlB321 as a carboxy [C]-terminal fusion to greenfluorescent protein (GFP) for possible future in vitro and in vivoimaging experiments. This construct was made by digesting the 800 bpInlB PCR product described earlier with SacI and BglII, and ligatingthis into the pRSETA-GFP plasmid, which places the InlB insert in-framewith the coding sequence of GFP (FIG. 4B). All constructs were doubledigested and evaluated by electrophoresis using a 1% agarose gel toconfirm the presence of the expected bands (FIG. 4C). Additionally, allthree constructs were sequenced to confirm their identity as well asverify that mutations were not introduced into the reading frames.

Recombinant Proteins InlB, InlB-PBK10 and GFP-InlB can be Produced inBacteria.

All three plasmid constructs described earlier (pRSET-InlB,pRSET-InlB-PBK10, and pRSET-GFP-InlB) were transformed into the E. colistrain, BLR (DE3) pLysS, for subsequent protein expression andpurification, as described in the Methods. This strain is more tolerantof repeat sequences in contrast to more traditional (i.e. BL21)expression strains, thus enabling the ability to obtain full-lengthproteins containing C-terminal polylysines. Meanwhile, the pRSET-Aplasmid encodes proteins as N-terminal fusions to a polyhistidinesequence. This histidine (His)-tag enables affinity purification usingnickel-chelating resin and provides an epitope for recognition by antiHis-tag antibodies.

All three constructs produced soluble proteins (InlB, InlB-PBK10, andGFP-InlB) in bacteria, which could be isolated by metal chelate affinitychromatography using nickel-conjugated resin, as described in theMethods. Anti-His tag antibodies recognized all three proteins, whichmigrated by denaturing gel electrophoresis at the predicted molecularweights of ˜37 kDa (InlB), ˜100 kDa (InlB-PBK10) and ˜63 kDa (GFP-InlB)(FIG. 5).

The Surface Level of c-MET Varies Among Different Tumor Cell Lines.

The first objective in characterizing InlB-PBK10 was to determinewhether this protein recognizes c-MET on tumor cells. Most of theliterature regarding c-MET levels associated with different tumor celllines is based either on total c-MET protein expression or RNAexpression, and thus is not informative with regard to the level ofc-MET displayed on the cell surface. Therefore, it was important tofirst determine the relative cell surface levels of c-MET on the panelof cell lines available for our use. This proved to be an initialchallenge because the majority of antibodies available for suchassessments have been generated against cytosolic domains of c-MET, andused to evaluate mechanisms of c-MET expression and activation. For thisstudy, the MET3 antibody, developed specifically against cell surfacec-MET, was used. We used a cell-surface ELISA to measure relativecell-surface levels of c-MET on a variety of cell lines. Briefly, thisapproach enables us to measure relative receptor levels on the surfaceon non-permeabilized cells and can be performed in a 96-well format,allowing both multiple replicates as well as the conservation ofprecious reagents. Our ELISA results show that the H1993 (lung cancercell line) and MDA-MB-231 (breast cancer cell line) are among the celllines with the highest surface levels of c-MET. RANKL (prostate cancercell line) and MDA-MB-435 (breast cancer cell line) display moderatelevels while LN-GFP (prostate cancer cell line) and Cos-7 (African greenmonkey kidney fibroblast) display low levels of cell surface c-MET (FIG.6).

The InlB-Derived Peptide Recognizes c-MET.

In order to assess the receptor-specificity of the targeting ligand, weused fluorescence activated cell sorting (FACS) to measure the relativelevel of InlB bound to c-MET positive cells. Two tumor cell lines weretested for the binding of InlB: one tumor cell line with high expressionof c-MET (H1993) and another tumor cell line with low expression ofc-MET (LN GFP). InlB exhibited a proportionately higher level of bindingto H1993 cells in comparison to LN GFP (FIG. 7A). To verify whether InlBis specifically bound to c-MET, we used a soluble peptide derived fromthe extracellular, ligand-binding domain of MET (MET peptide) as acompetitive inhibitor for InlB binding. An equimolar ratio of inhibitorto ligand predicts 50% reduction in receptor binding. In agreement,equimolar (1:1) concentrations of MET:InlB reduced InlB binding to H1993cells by 50%, indicative c-MET-selective binding. (FIG. 7B).

Cell-Binding by InlB-PBK10 Associates with c-MET Level and isCompetitively Inhibited by Free Ligand.

In the previous section, we showed that InlB has the capacity to bind toc-MET positive tumor cell lines through c-MET. The next goal was to testwhether receptor binding by InlB would be affected when embodied as partof a fusion protein. To evaluate this, we assessed the binding ofInlB-PBK10 to cell lines expressing high c-MET (MDA-MB-231) and lowc-MET (Cos-7) using cell surface ELISA. InlB-PBK10 showed a higher levelof binding to the cells with higher c-MET cell surface expression(MDA-MB-231) in comparison to cells expressing relatively low c-METlevels (Cos-7) (FIG. 7C). To further confirm that InlB-PBK10 recognizesc-MET, free InlB ligand was used as a competitive inhibitor. InlB-PBK10showed decreased binding to MDA-MB-231 cells in the presence ofincreasing concentrations of InlB, suggesting that InlB-PBK10 bindsthese cells through c-MET (FIG. 7D).

InlB-PBK10 Exhibits Binding to c-MET on Cells in Suspension.

As a further confirmation of c-MET recognition, we tested whetherInlB-PBK10 could bind to cells in suspension (in contrast to theprevious assay which assessed binding to adherent cells). We performed apull down assay in which we incubated the c-MET positive cell line,MDA-MB-435, in suspension with InlB-PBK10 in the presence of increasingconcentrations of free InlB ligand as a competitive inhibitor. Theconcentrations of free InlB ligand were chosen so that the molar ratiosof InlB-PBK10:InlB were 1:1, 1:5, and 1:10. InlB-PBK10 levelsco-precipitating with cell pellets were then assessed by Westernblotting using an antibody specific for InlB-PBK10 (Ad5 antibody, whichrecognizes the penton base). The levels of Inl-PBK10 binding decreasedas the concentration of InlB increased, with binding nearly completelyinhibited, thus verifying that InlB-PBK10 can recognize and bind c-METon cells in suspension (FIG. 7E).

InlB-PBK10 Internalizes into c-MET+ Cells.

To examine the survival and detection of InlB-PBK10 after cell uptake,we first incubated InlB-PBK10 with three different cell types,MDA-MB-231, MDA-MB-435 and H1993, at 4° C. to promote receptor bindingbut not internalization. To induce endocytosis, we then incubated thecells at 37° C. for the indicated times and fixed the cells at thespecific time points up to 30 minutes. Immunofluorescent staining andconfocal microscopy show that in all the three cell lines InlB-PBK10(shown in green) congregated on the cell membrane and gathered into fociat the cell membrane within the first 5 minutes of binding. InlB-PBK10then accumulated in the perinuclear region by 30 minutes. These datashow that InlB-PBK10 can internalize and accumulate inside the cellswithin 30 minutes after cell binding (FIG. 8). Cells were fixed at thespecific time points up to 30 minutes and imaged by laser scanningconfocal microscopy. Red, actin; Blue, nucleus. Bar, ˜10 microns.

InlB-PBK10 Delivers Toxic Molecules to c-MET+ Cells.

To evaluate the endosomolytic capacity of InlB-PBK10, we assessedwhether it could mediate the cytoplasmic entry of cytotoxic agents thatare inherently incapable of penetrating the cell membrane on their own.Sulfonated corroles containing gallium (III) (S2Ga or Ga-corrole) areintensely fluorescent compounds that can spontaneously assemble withproteins. These compounds cannot cross the cell membrane without amembrane-penetrating carrier to deliver them into cells to accesscytoplasmic targets of toxicity. 20-22 The Medina-Kauwe lab haspreviously shown that Ga-corroles alone are non-toxic but can promotecell death when delivered into HER2+ tumor cells by HerPBK10.

For this study, InlB-PBK10 was mixed with the Ga-corrole to promotenon-covalent assembly, and the resulting InlB-PBK10-Ga complexcharacterized by transmission electron microscopy (TEM) and dynamiclight scattering (FIGS. 9A, and 9B). TEM (right panels of FIG. 9A) showsthe spherical assemblies formed when Ga-corrole (+S2Ga) is added toInlB-PBK10 compared to InlB-PBK10 alone (−S2Ga). The imaging shows thatthe protein and corrole form 10-20 nm diameter clusters. Dynamic lightscattering, used to measure particle size, shows that InlB-PBK10 aloneforms particles of 8.4 nm average diameter while InlBPBK10-Ga is about16.4 nm diameter.

To examine whether InlB-PBK10-Ga could penetrate cells and inducecorrole-mediated toxicity in c-MET positive cancer cell lines, weexposed MDA-MB-435 cells to 1 μM, 5 μM and 10 μM of each of thefollowing: InlB-PBK10, S2Ga and InlB-PBK10-Ga. At 1 μM concentration,InlB-PBK10-Ga reduced cell survival by 60% whereas 1 μM and higherconcentrations of S2Ga alone and InlB-PBK10 alone did not affect cellsurvival. These data affirm that InlB-PBK10 bears endosomolyticcapacity, and shows that this construct is capable of deliveringcytotoxic agents such as Ga-corroles into cells expressing cell surfacec-MET (FIG. 9C).

To further evaluate the therapeutic capacity of InlB-PBK10, it wasassessed whether this protein could be used to deliver more mainstreamchemotherapeutic molecules. Doxorubicin (Dox) is an FDA approvedcytotoxic agent that is used for a broad range of cancer types. If suchdrugs could be targeted mainly to tumor cells, it would be beneficial interms of reducing side effects. In this approach, first Dox isintercalated into a double stranded oligo to form DNA-Dox, which thencan bind to the polylysine of InlB-PBK10 (via charge interactions withthe DNA phosphate backbone) and form a complex that we have designated,I-Dox. The I-Dox complex (5 μM with respect to Dox concentration) wasincubated on the indicated cell lines under the conditions described inthe Methods. InlB-PBK10 was incubated at equivalent concentrations tothat in the I-Dox complex. Cell survival was assessed by metabolic (MTT)assay at 24 h after treatment. The I-Dox complex induced significanttoxicity to cells with high c-MET expression (H1993) when compared tocells with low c-MET expression (LAPC4) (FIG. 9D). H1993 cells werefirst treated with free InlB ligand to block c-MET, followed by exposureto I-Dox at the same conditions as above. InlB-PBK10 alone appeared toslightly but not significantly reduce cell numbers, suggesting that themain mechanism of cell death was through the delivery of Dox.Additionally, free InlB ligand inhibited cytotoxicity by I-Dox,suggesting that I-Dox-mediated cytotoxicity occurs through c-MET bindingand entry for the delivery of Dox (FIG. 9E).

Experimental Procedures

Cells.

Human breast cancer cell lines (MDA-MB-435* and MDA-MB-231) wereobtained from the National Cancer Institute. Human lung cancer (H1993),and African Green Monkey Kidney Fibroblast (Cos-7) cell lines wereobtained from ATCC. Human ovarian cancer cells (A2780) were obtainedfrom Sigma-Aldrich. Prostate cancer lines (LNCaP^(Neo/RANKL),LNCaP^(Neo)) were kindly provided by Dr. Leland Chung (Cedars-SinaiMedical Center). MDA-MB-435 and MDA-MB-231 were maintained in DMEM, 10%v/v fetal bovine serum (FBS, Sigma-Aldrich), and 1% v/vpenicillin/streptomycin (Sigma-Aldrich) under 5% CO₂. H1993 and A2780cells were maintained in RPMI, 10% v/v FBS, and 1% v/vpenicillin/streptomycin under 5% CO₂. Cos-7 cells were maintained inDMEM/F12 medium, 20% v/v non-heat inactivated fetal bovine serum (ATCC30-2020), and 1% v/v penicillin/streptomycin (Sigma-Aldrich) under 5%CO₂. LNCaP^(Neo/RANKL), LNCaP^(Neo) were maintained in RPMI medium, 10%v/v FBS, and 1% v/v penicillin/streptomycin and 200 μg of G418 disulfatesalt solution (Sigma Aldrich G8168). 200 μg/ml of hygromycin (GEMINIBio-products 400-123) was added to the medium of the LNCaP^(Neo/RANKL)cells to maintain the RANKL-expression plasmid. In order to maintainequivalent confluency between the cell lines, in certain assays theywere plated according to their growth rate.

DNA Constructs.

The targeting construct, InlB-PBK10, was produced by a two-step cloningmethod (Summarized in FIG. 8A) in which sequences encoding InlB andPBK10 were sequentially ligated together into the protein expressionplasmid, pRSET-A (Invitrogen, Carlsbad, Calif., USA). A plasmidconstruct encoding aa 36-321 of internalin B (pMET-30-InlB321)(CeBiTec), Bielefeld University, Germany) was used as a template forpolymerase chain reaction (PCR) amplification using forward and reverseoligonucleotide primers containing the sequences,5′-AGTGAGCTCGAGACTATCACTGTG-3′ (SEQ ID NO:1) and5′-GTTGGTGACTTTCTCCCACCTTCACCACCTTCATCTAGATATCCATGGTAT-3′ (SEQ ID NO:2)respectively. A SacI restriction site was introduced in the forwardprimer for in-frame insertion into pRSET-A. BglII and KpnI restrictionsites were introduced into the reverse primer for subsequent in-frameinsertion of PBK10 just 3′ to the InlB coding sequence. The reverseprimer also contains a sequence encoding a flexible linker(GlyGlySerGlyGlySer) (SEQ ID NO:3) in-between the InlB and PBK10sequences. The 800 bp InlB PCR product was digested with SacI and KpnIfor ligation into pRSET-A. The resulting construct, pRSETA-InlB, wasthen digested with BglII and HindIII to accommodate the insertion ofPBK10 in-frame with InlB. PBK10 was excised from the plasmid,pRSET-PBK10, using BamHI and HindIII restriction enzymes, and insertedinto the BglII-HindIII sites of pRSETA-InlB, resulting in thepRSET-InlB-PBK10 construct.

The pRSETA-GFP-InlB construct that encodes InlB321 as a carboxy[C]-terminal fusion to green fluorescent protein (GFP) was made bydigesting the 800 bp InlB PCR product, described earlier, with SacI andBglII, and ligating this into the pRSETA-GFP plasmid for in-framecloning of InlB321.

Protein Expression and Purification from Bacteria.

Overnight cultures of BLR(DE3)pLysS (Novagen, Madison, Wis., USA)bacterial transformants were inoculated 1:50 in LB containing 0.5 mg/mlampicillin and 0.034 mg/ml chloramphenicol, and 0.0125 mg/mltetracycline. When cultures reached an absorbance reading of 0.6 at anoptical density wavelength of 600 nm (OD 600), cultures were inducedwith 0.4 mM IPTG and grown for a further 3 h at 37° C. with shaking.Cultures were harvested and pelleted. Cell pellets were resuspended inlysis buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 2 mM EDTA, pH 8.0) andlysed by addition of 0.1% Triton X-100 and one cycle of freeze-thawing,with 1 mM phenylmethylsulfonyl fluoride (PMSF) added during the thaw.After thawing, lysates received 10 mM MgCl₂, and 0.01 mg/ml DNase andlysates were rocked for 10 minutes at room temperature to allowdigestion of genomic DNA before lysates were returned to ice, afterwhich they received 300 mM NaCl and 10 mM imidazole. Lysates weretransferred to pre-cooled centrifuge tubes, balanced, and centrifuged at4° C. in a pre-chilled rotor at 39,000×g for 1 hour. Supernatants wererecovered, added to Ni-NTA resin (Qiagen, Valencia, Calif., USA)pre-equilibrated with MCAC-10 (50 mM NaH₂PO₄, 300 mM NaCl, 10 mMimidazole, and 0.1% Triton X-100, pH 8.0), and incubated for 1 h on ice.The resin containing bound protein was washed with 20 mL of MCAC-10buffer one time for 10 minutes rocking on ice and three times withMCAC-20 (50 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, and 10% glycerol,pH 8.0), followed by elution with 2 mL of a solution of 50 mMNa-phosphate, pH 8.0, 300 mM NaCl, 250 mM imidazole, and 10% glycerol.Proteins were simultaneously buffer exchanged into low-salt buffer andconcentrated by ultrafiltration (Amicon Ultra CentrifugalFilters—Ultracel—50K (Millipore, Bedford, Mass., USA) and theirconcentrations measured using the BioRad protein quantification assay(BioRad Laboratories, Hercules, Calif., USA).

Protein Detection.

Denaturing polyacrylamide gel electrophoresis was performed in adiscontinuous gel buffer system as known in the art. Proteins wereelectrically transferred on to nitrocellulose using 192 mM glycine, 25mM Tris, and 20% methanol in a BioRad semi-dry transfer cell set atconstant voltage (20 V) for 40 min. Blots were blocked with 3% bovineserum albumin in Tris-buffered saline (10 mM Tris, pH 7.5, 150 mM NaCl).Blots were incubated overnight with anti-RGS-His Tag antisera (Qiagen)at a 1:1500 dilution in blocking buffer. Antibody-antigen complexes weredetected by incubation with horseradish peroxidase (HRP) conjugatedsecondary antibodies (Sigma, St Louis, Mo., USA), followed by reactionwith HRP substrate and chemiluminescence detection reagents (ThermoFisher), and exposure to film (Hyperfilm ECL; Amersham PharmaciaBiotech).

ELISA Based Assay for c-MET Cell Surface Expression.

To determine c-MET levels on cell lines, cells were plated in 96-wellplates using the following cell numbers: 8×10³ MDA-MB-231, MDA-MB-435,A2780, LNCaP^(Neo/RANKL), LNCaP^(Neo) and 9×10³ for H1993 and LAPC4 perwell. 48 Hrs later media was aspirated and cells were briefly washedwith phosphate-buffered saline (PBS) containing 1 mM MgCl₂ and 1 mMCaCl₂ (PBS+), then fixed in 4% PFA in PBS for 12 min at room temperature(RT), followed by 3 washed with PBS+ (200 μL per well) before a 1 hrincubation in blocking solution (3% BSA/PBS, 100 μL per well) at RT.Anti-c-MET antibody (mouse monoclonal anti-c-MET used at 0.87 μg/mL; Dr.Knudson) was added in triplicate wells (100 μL per well) and incubatedfor 1 hr at RT. Cells were washed three times with PBS+ and incubatedfor 1 hr at RT with HRP-conjugated secondary antibody at a 1:2000dilution. Cells were washed 3 times with PBS+ and once with distilledwater, and 100 μL of TMB (eBioscience) solution was added to each well,according to manufacturer's instructions. The plates were incubated withsubstrate for 30 min (or until the blue color development was visible)in the dark, and the reaction was stopped by adding 100 μL of 1N HCl.Absorbance were measured at 450 nm in a Spectra MaxM2 plate reader(Molecular Devices Corp.) A crystal violet assay was then performed todetermine relative cell number. Briefly, cells were washed once withPBS+ (200 μL/well) and incubated with 0.1% crystal violet (100 μL/well)for 15 minutes at RT. Cells were thoroughly washed 4 times with PBS+(200 μL/well) and incubated with 95% Ethanol (100 μL/well) for 10minutes at RT. Absorbances were measured at 490 nm.

Cell Binding Assay.

To determine the binding of different concentrations of InlB-PBK10 tocells, cells were plated in 96-well plates at the followingconcentrations: 8×10³ LNCaP^(Neo/RANKL), LNCaP^(Neo) and 9×10³ for H1993and LAPC4 per well. At 48 hrs later, media was aspirated and cells werebriefly washed with 100 μL Buffer A (serum free DMEM, 20 mM HEPES pH7.4, 2 mM MgCl₂, 3% BSA). Cells were incubated with 50 μL of buffer Acontaining the indicated concentrations of InlB-PBK10 for 1 hour on icewith agitation at 4° C. The cells were washed with PBS+ (200 μL/well)one time and subject to cell surface ELISA as described earlier (cellsurface expression ELISA for c-MET), with the following modifications:To detect InlB-PBK10, plates were incubated overnight with primaryantibody (RGS-His; Qiagen) at 1:1500 dilution, and one hour at roomtemperature with secondary antibody (goat anti mouse, at 1:2000dilution).

For competitive inhibition assays, the indicated concentrations of InlBwere incubated on cells before adding InlB-PBK10. Specifically, celllines plated as described earlier were briefly washed with 100 μL BufferA, followed by incubation in 50 μL of buffer A containing 1 μM, 5 μM or10 μM of InlB for an hour on ice with agitation at 4° C. Cells werewashed one time with Buffer A (100 μL/well) and then incubated with 50μL of Buffer A containing 1 μM of InlB-PBK10 for an hour on ice withagitation at 4° C. The cells were washed with PBS+ (200 μl/well) onetime and processed for immune-detection of surface-bound InlB-PBK10 asdescribed earlier, except plates were incubated with an antibody thatrecognizes the penton base domain of InlB-PBK10 (Ad5 antibody; Abcam) at1:5000 dilution. Plates were incubated with secondary antibody (goatanti rabbit, at 1:2000 dilution), for one hour at room temperature.Binding was detected as explained earlier for c-MET cell surfaceexpression ELISA.

Cell Survival Assay.

MDA-MB-435 cells were plated in 96-well plates at 8×10³ cells per well.48 hrs later medium was aspirated and the cells were incubated with30-50 μl of media containing the different concentrations ofInlB-PBK10-Ga and S2Ga (1, 5 and 10 μM) for 4 hours on a rocker at 37°C. in a 5% CO₂ incubator. After the 4 hour incubation period, additionalmedia was added to bring the total volume per well to 100 μL and cellswere incubated for approximately 24 hrs without rocking. PromegaCellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay was usedto measure cell viability. Medium was removed and 100 μl fresh media wasadded to the wells. Per manufactures' instructions, 2.0 mL of MTSsolution was mixed with 100 μL of PMS solution and 20 μL was added toeach well. Plates were incubated at 37° C. with 5% CO₂ and absorbancewas read at 490 nm at 1 and 2 hours after the addition of the MTTreagents. Crystal violet staining was then performed to determine therelative cell number. Briefly, cells were washed with PBS+, containing 1mM Ca²⁺ and 1 mM Mg²⁺, then incubated with 0.1% crystal violet(1000/well) for 15 minutes at RT and then washed 4 times with PBS+ (200μL/well). Plates were incubated with 95% Ethanol (100 μL/well) for 10min. at RT, and absorbance was measured at 590 nm.

InlB-PBK10 Uptake/Intracellular Trafficking Assays.

For InlB-PBK10 uptake assays, cells were plated on coverslips in atwelve-well dish at 1×10⁵ cells/well and grown for 2 days. On the day oftreatment, dishes were transferred on ice and the cells were treated forthe indicated experiments according to the following methods. To studythe time course of uptake, cells were washed twice with cold PBS, andthen 0.4 mL of Buffer A with 8 μg of InlB-PBK10 was added to each well.Dishes were incubated for 1 h on ice rocking at 4° C. to promotereceptor binding but not internalization, and then cells were washedwith cold PBS to remove unbound protein. Wells then received pre-warmedcomplete media, and dishes were incubated at 37° C. at 5% CO₂ for theindicated time points. The individual coverslips were then removed,washed three times with PBS/1% MgCl₂ then then fixed with 4%paraformaldehyde in PBS for 15 min at room temperature. Coverslips werewashed three times with PBS, then incubated with 50 mM ammonium chloridein PBS for 5 min, 0.1% Triton X-100 in PBS for 5 min and then blockedwith 1% BSA in PBS for 30 minutes at room temperature. Cells oncoverslips were incubated with primary antibodies overnight at 4° C.,washed three times with PBS and incubated with secondary antibodies for1 h at room temperature in the dark. Where indicated, cellscounterstained for actin and nuclei were incubated with Texas Redx-Phalloidin (Invitrogen T7471) at 1:100 dilution during the secondaryantibody incubation, followed by a 5 min incubation in DAPI at 300 nMfinal concentration. Phalloidin, DAPI and primary and secondaryantibodies were all diluted in 1% BSA in PBS. After secondary antibodyand DAPI treatment, cells on cover slips were washed three times withPBS and mounted in Prolong Antifade mounting medium (Molecular Probes,Eugene, Oreg., USA). For detecting InlB-PBK10 the RGS-His antibody(Qiagen) was used at 1:150 dilution and a fluorophore-conjugated goatanti-mouse (FITC-conjugated) at dilution of 1:500 was used forfluorescence detection at 488 nm for InlB-PBK10, 405 nm (DAPI) fornuclei and 532 nm (Texas red) for F-Actin.

InlB-PBK10-Dox (I-Dox) Assembly.

The viral capsid-derived fusion protein, InlB-PBK10, was assembled withdoxorubicin following well-established procedures. Briefly,complementary oligonucleotide duplexes were prepared by mixing togetherwith equal molar concentration of the 30 base oligonucleotide, LLAA-5(5′-CGCCTGAGCAACGCGGCGGGCATCCGCAAG-3′) (SEQ ID NO:4) and itscorresponding reverse complement, LLAA-3. The mixture was boiled for 5minutes and cool down to room temperature for 30 min to promoteannealing of oligonucleotides. The double stranded oligos were mixedwith Dox at 1:10 molar ratio (DNA:Dox) and incubated at room temperature(RT) for 30 minutes, followed by incubation with InlB-PBK10 at 6:1 molarratio of InlB-PBK10:DNA-Dox in HBS, and ultrafiltration using 50 k MWcutoff (mwco) filter membranes (which was prepared by pre-incubatingwith 10% glycerol for 2 hours-overnight) to isolate I-Dox fromincompletely assembled components. The complexes were centrifuged at4000×g for 15 min or until volume was reduced by ≥80%. The Doxconcentration in I-Dox was determined by extrapolating the measuredabsorbance at 480 nm or fluorescence at 590 nm (Ex: 480 nm) against aDox absorbance or fluorescence calibration curve (SpectraMax, MolecularDevices, USA). The concentration of Dox was calculated by applying theBeer-Lambert equation: (Absorbance at λmax/Dox extinctioncoefficient)×dilution factor=concentration (M), using a Dox coefficientof 11, 500 M⁻¹ cm⁻¹. The I-Dox doses used in experiments is based on theconcentration of Dox in I-Dox.

InlB-PBK10-Ga Assembly.

InlB-PBK10 was noncovalently assembled with sulfonated gallium corrole(S2Ga) by incubating a 10× molar excess of corrole with InlB-PBK10 inthe dark at 4° C. for 1 h with gently agitation. The unbound corroleswere removed by ultrafiltration through 50 K MW cutoff spin columnfilters (Millipore Corporation, Billerica, Mass., USA) according to themanufacturer's procedures for filtration, and the complexes washed withPBS until the filtrate clarified. The retentates retained their brightgreen color (indicative of corrole pigment) throughout the filtrationprocess. The retentates were resuspended in PBS and measured forabsorbance at the λ_(max) of the corrole to obtain the corroleconcentration as described earlier. Whereas the λ_(max) of S2Ga, forexample, shifts from 424 to 429 nm when bound to proteins, this does notdramatically change the estimation of corrole concentration incomplexes.

FACS Analysis.

To evaluate binding of InlB321, H1993 and Ln GFP cells were cultured for48 hrs. After washing two times with PBS, the cells were detached byincubating in 2 mM EDTA for 5-10 min at 37 C. PBS containing 0.01% MgCl₂and CaCl₂ (0.01% PBS+) was added to the cells and spun for 4 min at 2000rpm. Cells were washed with 0.01% PBS+ three more times, resuspended,counted, and divided into the Eppendorf tubes. The tubes were spun for 2minutes at 300×g and resuspended with 3% milk in PBS containing theindicated concentrations of InlB and incubated on ice in the cold roomfor 1 hr. Cells were then washed four times with PBS and incubated withRGS-His antibody diluted 150 fold in 3% BSA in PBS for 30 min at roomtemperature. Cells were washed four times before re-suspending them withsecondary antibody antibody, Alexa Fluor 647 Goat Anti-Mouse IgG (H+L),in 3% BSA and incubated at room temperature for 30 min. Cells werewashed 4 times, incubated with 0.1% PFA for 15 min and then washed againfor four times and analyzed with Beckman Coulter Cyan ADP FACS machine.

To verify that cell binding by InlB321 is through c-MET, InlB321 and METpeptide (YCP2247 SPEED BioSystems. Rockville, Md.) were incubated in 3%milk in 1×PBS for 30-40 min on ice before binding to cells. The bindingassay was performed as described above.

Cell Pull Down Assay.

MDA-MB-435 cells grown for 48 hrs were lifted using 2 mM EDTA in 1×PBSat 37° C. with agitation for 5-10 min. After spinning the cells for 5min at 300×g, they were washed once and resuspended in Buffer A+3% BSAand divided into four Eppendorf tubes (2×10⁶ cells/tube). The cells werespun again and resuspended into 500 μL of buffer A+3% BSA. In three ofthe tubes 0.4 μM, 2 μM and 4 μM of InlB was added and no protein wasadded to the fourth tube; all tubes were incubated on ice for 1 hr.Following washing and spinning of all cells to remove unbound InlB, 0.4μM of InlB-PBK10 in 500 μl of Buffer A was added to all four tubes,followed by rocking on ice for 2 hours to promote receptor binding butnot internalization. Cells were washed and spun three times as describedearlier. Pellets then suspended in 50 μL of SDS-PAGE sample buffer andevaluated by SDS-PAGE/Western blotting using an antibody specific toInlB-PBK10 (Ad5 antibody, which recognizes the penton base) at the1:5000 dilution.

Example 3: Inl-PBK10 Biodistribution in Nu/Nu Mice Bearing BilateralFlank MDA-MB-435 Tumors

Background

As shown in the previous example entitled “A Protein Nano-ConstructTargeted to c-Met”, FIG. 6, MDA-MB-435 cells display marked levels ofc-MET on the cell surface. Moreover, as shown in FIG. 7E, the c-METtargeted protein construct, InlB-PBK10, binds these cells through aninteraction that is competitively inhibited by the c-MET ligand, InlB,indicating that this construct can bind these cells through c-MET. FIG.8 shows that InlB-BK10 enters these cells after receptor binding, andFIG. 9C shows that the protein can mediate the delivery of a membraneimpermeable toxic molecule (gallium-metallated corrole) into thesecells, causing cell death (thus also verifying that Inl-PBK10 is capableof endosomal membrane disruption).

Results

Here we evaluated the biodistribution of Inl-PBK10 in a mouse withMDA-MB-435 tumors to assess whether it is capable of accumulating atthese tumors in vivo. To do so, a female nu/nu mouse bearing bilateralflank xenografts of MDA-MB-435 tumors received a single tail veininjection of Alexa680-labeled InlB-PBK10 (2 nmol protein) and was imagedby Xenogen imaging at the indicated time points after injection, using640 nm excitation and 700 nm emission filters. Whole animal imagingshows considerable clearance of fluorescence from the animal by 4 hafter injection (FIG. 10A). After the 4 h time point, the mouse wassacrificed and tumors and tissues removed for further imaging. While thetumors show some fluorescence accumulation, a significant proportion ofinjected material was eliminated to the kidney by 4 hours while somefluorescence could be detectable in the liver (FIG. 10B). Remainingtissues, including heart, spleen, lung, brain, and skeletal muscle,showed no detectable fluorescence (FIG. 10B). Further studies willentail tissue harvest at earlier and later time points to determinewhether delivery to the kidney is the result of rapid elimination, aswell as determining biodistribution with a competitive inhibitor toverify that tumor delivery occurs via c-MET.

Example 4: Nanobiologic Targeting of Brain Metastatic Breast Tumors

Elevated cell surface levels of the human epidermal growth factorreceptor subunit 3 (HER3) is associated with metastatic breast tumors,including those that spread to the brain. Whereas a number of targetedtherapies are currently used to combat peripheral breast tumors, thedelivery of these molecules to brain metastases is limited by the bloodbrain barrier (BBB). This is exemplified by HER2+ breast tumors thatmetastasize to the brain: these tumors, while targetable outside of thecentral nervous system (CNS) by HER2 antibodies such as trastuzumab, areunreachable by these same antibodies because the HER2 subunit, thoughpresent on the brain endothelium, does not mediate antibody transcytosisacross the blood vessel wall.

HER3, on the other hand, undergoes rapid transcytosis across the brainendothelium upon ligand binding, which normally occurs to mediate thedelivery of neuregulin growth factors for neural growth and maintenance.We have developed a self-assembling nanobiological particle, HerMn,which uses HER3 as a portal for targeted entry of toxic molecules intotumor cells. HerMn is a 10-20 nm diameter serum-stable particlecomprised of the receptor-targeted cell penetration protein, HerPBK10,non-covalently assembled with a sulfonated manganese(III) corrole (S2Mnor Mn-corrole) (FIG. 11). The targeting domain of HerPBK10 is derivedfrom the ligand, heregulin alpha, which specifically interacts with thehuman epidermal growth factor receptor subunit 3 (HER3) and inducesrapid receptor-mediated endocytosis. As HER3 is the preferreddimerization partner of HER2, and HER2-3 heterodimers are prevalent onHER2+ tumor cells, we have previously shown that HerPBK10 can targettherapeutic and imaging molecules to HER2+ tumors in mice, mediatingtumor growth ablation at >10× lower dose compared to the chemotherapyagent, doxorubicin, while sparing heart and liver tissue, and with nodetectable immunogenicity. Tumor-targeted toxicity by HerMn occurs bymitochondria membrane disruption and superoxide-mediated damage to thecytoskeleton. HerMn can also elicit tumor-selective detection bymagnetic resonance imaging (MRI) due to the paramagnetic property of thecorrole.

HerMn appears to distribute to the brain after systemic injection inmice, in addition to showing preferential homing and toxicity tosubcutaneous HER2+ tumors. Interestingly, the Mn corrole is known toexhibit neuroprotective effects due to its antioxidant activity onnormal tissue. In support, HerMn supports human cardiac cell survival exvivo. Taken altogether, it is intriguing to speculate that HerMn mayhave the capacity to target toxicity to brain-metastatic breast tumorswhile sparing off-target tissue due to both its targeting capacity andability to provide beneficial protective effects to normal tissue suchas the brain and heart.

Background

Brain Metastasis is a Serious Clinical Problem.

Patients with breast cancer metastases to the brain on average surviveless than one year. While a growing repertoire of targeted therapieshave emerged for treating peripheral tumors, most are unsuitable fortreating brain metastases due to their inability to cross the bloodbrain barrier (BBB). An example of this is the use of the monoclonalantibody, trastuzumab (Tz), which is directed against the humanepidermal growth factor receptor subunit 2 (HER2) for treating HER2+breast cancer. HER2+ cancers are associated with aggressive tumors,recalcitrance to standard therapies, metastasis, and increasedmortality. HER2+ tumors that metastasize to the brain cannot be treatedwith Tz because HER2, though present on the brain endothelium, does nottranscytose across the blood vessel wall. Triple-negative breast cancer(TNBC), which is HER2− and also brain-metastatic, has even fewer optionsdue to the lack of specific cell surface biomarkers. While lapatinib(Lp), a small molecule tyrosine kinase inhibitor (TKI) of HER2 and EGFR,can readily permeate the cell membrane to target both tumor types, thesetumors are likely to resist such inhibitors due in part to elevation ofHER3.

Results

HerPBK10 is Specific to HER3.

HerPBK10 contains the receptor binding region of the HER3 ligand,heregulin-α (amino acids 35-239, comprising the Ig-like and EGF-likedomains), fused to a membrane-penetrating moiety derived from theadenovirus penton base capsid protein (FIG. 11A). HerPBK10 wasoriginally designed as a non-viral gene transfer vector, containingpayload assembly, targeting, internalization, and endosomal penetrationfunctions within a single multidomain protein molecule. As HER3 is thepreferred dimerization partner of HER2, and HER2-3 heterodimers areprevalent on HER2+ tumor cells, it has been shown that HerPBK10 cantarget therapeutic and imaging molecules to HER2+ tumors in mice,mediating tumor growth ablation at >10× lower dose compared to thechemotherapy agent, doxorubicin, while sparing heart and liver tissue,and with no detectable immunogenicity.

The specificity of HerPBK10 to HER3 is supported by its ability to bindto an immobilized peptide containing the extracellular domain of humanHER3 (FIG. 12A). This is inhibited by pre-adsorption of HerPBK10 withfree HER3 peptide in vitro (FIG. 12A). The same peptide also inhibitsbinding to both human (FIG. 12B) and mouse (FIGS. 13B-C) HER3+ cells.Notably, the ligand-binding domain of mouse and human HER3 share high(94%) sequence identity (FIG. 13A). While HER2-3 heterodimers areprevalent on HER2+ tumor cells, the heterodimerization-inhibitingantibody, pertuzumab (Pz), does not prevent HerPBK10 binding to cellularHER3 (FIG. 12B), indicating that HER2-3 dimerization is not required forHerPBK10 binding. Binding is also not inhibited by a HER4 peptide orbetacellulin (which blocks HER4) (FIG. 12B), indicating that HerPBK10 isHER3-specific.

Sera from HER2+ patients and age-matched controls do not preventHerPBK10 binding to HER2+ cells in culture (and show no significantdifferences between one another), in contrast to the addition of excessrecombinant ligand (+Her) used as a competitive inhibitor (FIG. 12C).Previous studies have shown that repeat dosing of HerPBK10 inimmune-competent mice do not produce detectable neutralizing antibodyformation against the protein. Moreover, polyclonal antibodies generatedagainst whole adenovirus that can recognize HerPBK10 do not preventreceptor binding to cells in culture.

HerPBK10 Self-Assembles with Mn-Corroles to Form HerMn Nanoparticles.

The carboxy [C]-terminus of HerPBK10 is comprised of a decalysine tail(FIG. 11A), which can mediate electrophilic binding to anionicmolecules, including corroles. Corroles are macrocyclic molecules withstructural similarity to porphyrins and likewise can contain a metalligand (FIG. 11B). Sulfonated corroles are amphiphilic, soluble inphysiological solutions, and can spontaneously bind proteins throughnon-covalent interactions, including electrophilic and hydrophobicinteractions. The anionic sulfonato groups prevent non-specific cellentry due to repulsion by the negatively charged cell membrane, thusenabling the potential to direct corrole delivery into target cells viaa carrier protein. We have combined HerPBK10 with a sulfonatedmanganese(III) corrole (S2Mn or Mn-corrole) to form rapidlyself-assembling 10-20 nm diameter particles designated HerMn (FIGS.11C-D). These particles contain multiple corrole molecules bound to asingle protein (25-35 corroles/protein), and can withstand high-speedultrafiltration. These findings are consistent with our previous studiesand those of our collaborator showing that corroles can bind in proteinpockets with negligible dissociation, and once bound to HerPBK10, resisttransfer to serum proteins.

HerMn Targets HER3+ Tumors.

HerMn exhibits targeted toxicity to HER2+/HER3+ but not HER2−/HER3−tumor cells in culture while Mn-corrole has no effect on cell survival.The data is shown in FIG. 14, where each cell line received theindicated concentration of HerMn or S2Mn and was assessed for survival24 hours later via crystal violet (CV) stain. N=3 per concentrate, from3 separate experiments. It has been shown that the targeting ligand ofHerPBK10 induces rapid endocytosis after receptor binding. As sulfonatedcorroles are unable to breach the endosomal membrane on their own, thepenton base moiety of HerPBK10 enables effective membrane disruptionafter endocytic uptake, and entry into the cytoplasm.

In one experiment, cells received 10 μM S2Mn or HerMn, then TMRM (30 nM)in HBSS 24 hours later. The results are shown in FIG. 15A, where thecontrol is PBS-treated. Confocal fluorescence images, FIG. 15B, showsuperoxide-mediated collapse of actin (red) and tubulin (green) by HerMn(5 μM) after 24 h incubation on MDA-MB-435 cells. S2Mn (5 μM), HerPBK10(at equivalent protein concentration as HerMn) and PBS served ascontrols. Additional cells received Tiron (5 mM) for 1 h before HerMntreatment. Blue, nucleus. Scale bar=10 μm

Once in the cytoplasm, HerMn collapses mitochondrial membrane potential(FIG. 15A) and the cytoskeleton through superoxide elevation andoxidative damage to these structures (FIG. 15B), consistent withgallium(III) corrole (S2Ga or Ga-corrole).

It is discovered that Ga-corrole directly binds the mitochondrial outermembrane protein, TSPO. The results are shown in FIG. 16. Solublerecombinant TSPO protein was incubated with S2Ga at equivalent molarconcentrations (1 μM) for ˜20 min at room temp followed byultrafiltration to remove free, unbound S2Ga. Retentates were evaluatedfor the presence of TSPO protein-bound corrole by measuring theabsorbance and fluorescence spectra. Where indicated, PK11195 was usedas a competitive inhibitor for the porphyrin-binding site on TSPO (FIGS.16A-B). There is evidence of HerGa interaction with TSPO in situ.MDA-MB-435 cells were transfected with a plasmid expressing exogenousTSPO (as a competitive inhibitor of endogenous TSPO binding) 24 h beforecells were treated with HerGa and examined for HerGa-mediatedmitochondrial disruption, evidenced by reduced red fluorescent dyeaccumulation in mitochondria and accumulated green fluorescence in thecytoplasm (FIGS. 16C-D).

TSPO translocates porphyrins and other metabolites into mitochondria forprocessing, interacts with components of the mitochondrial permeabilitytransition pore complex, and contributes to cellular homeostasis.Ga-corrole specifically recognizes the porphyrin-binding site on TSPO(FIGS. 16A-B), as corrole binding can be competitively inhibited byPK11195, which inhibits porphyrin binding to TSPO. Overexpression ofrecombinant soluble TSPO in MDA-MB-435 cells prevents corrole-mediateddisruption of mitochondrial membrane potential (FIGS. 16C-D), suggestingthat the corrole interacts with TSPO in situ. As the Mn-corrole exhibitssimilar mitochondrial membrane disruption as the Ga-corrole, it islikely that TSPO is a molecular target of Mn-corrole. In a xenograftmouse tumor model, HerMn homes to tumors in vivo after systemicdelivery, bypassing most normal tissue including the heart (FIG. 17),and ablates tumor growth at very low pharmacologic dose (0.008 mg/kg)(FIG. 18A). The Mn-corrole is also paramagnetic, thus useful for MRI.

It has been found that, in addition to accumulating in tumors aftersystemic delivery in mice, HerMn can distribute to the brain in contrastto trastuzumab (Tz) (FIG. 17). HerPBK10 injected in the tail veins ofmice without tumors also show brain localization, whereas systemicdelivery of PBK10 (which lacks the HER3 targeting ligand) shows nodetectable brain delivery (FIG. 19). It has also been found that HerMnis not only non-toxic to human cardiosphere-derived cells (CDCs), but atescalating doses with extended exposure, augments CDC survival inculture, in contrast to doxorubicin (which has known cardiotoxicity),and HerPBK10 alone, which has no toxic or growth promoting effect ontumor cells or CDCs (FIG. 18B). This supports previous findings in amodel of optic neuropathy in vitro and in vivo, showing that theMn-corrole can be neuroprotective. This appears to be unique to theMn-corrole, as the Ga-corrole showed no such neuroprotective effect.

Together, these findings indicate that HerMn imparts a beneficial effecton normal tissue while targeting toxicity to tumor tissue, especiallybrain metastases with elevated HER3. The targeting specificity of HerMnrenders this point less relevant, as a minority of systemic particlesappear to distribute to non-tumor tissue (FIG. 17). But, as relativelylow corrole levels afford neuroprotection, as well as tumor toxicity(FIG. 18A), this potential dual activity is worth exploring.

Applications

HerMn has the capability of delivering toxicity to brain-metastaticbreast tumors while sparing off-target tissue due to both its targetingcapacity and ability to provide protection to normal tissue such as thebrain and heart.

Example 5: Targeting Beta-1 Integrins Using Yersinia enterocoliticaInvasin-Derived Peptide

Yersinia enterocolitica is a bacterial pathogen that invades theintestinal epithelium, particularly the Peyer's patches of theintestinal wall, and causes food poisoning. Binding and entry ofintestinal epithelia occurs through the interaction of the bacterialinvasin (Inv) protein with beta-1 integrins, which are predominantlyexpressed on epithelial cells overlying Peyer's patches.

The plasmid containing the nucleotide sequence encoding Invasin(pHIT123-Inv) was used as a PCR template, and oligonucleotide primerswere designed to amplify the minimal sequence encoding the beta-1integrin binding site (˜600 bp) while introducing restriction sites forin-frame cloning into exogenous peptides for targeted delivery. Theprimers used included the following sequences:5′-ACAGAGCTCATAACCGGCATTAACGTGAAT-3′ (SEQ ID NO:6);5′-CTGTGTGCGGAGCCGCAATAGGAATTCATC-3′ (SEQ ID NO:7); and5′-GATGAATTCCTATTGCGGCTCCGCACACAG-3′ (SEQ ID NO:8). These primers areused for amplifying and adding SacI and EcoRI restriction sites to Invinsert. Additional primers included the following sequences:5′-GTCCAAAACCAAGAAAAGGCGGAGCAGCTGTAC-3′ (SEQ ID NO:9);5′-ACAATGACGCGTGTACAGCTGCTCCGCCTTTTCTTGGTTTTGGAC-3′ (SEQ ID NO:10);5′-CCGCTGTGTGCGGAGCCGCAAGGAGGAACGCGTACACAC-3′ (SEQ ID NO:11);5′-GTGTGTACGCGTTCCTCCTTGCGGCTCCGCACACAGCGG-3′ (SEQ ID NO:12); and5′-ACACACGGATCCTTGCGGCTCCGCACACAGCGG-3′ (SEQ ID NO:13). These primersintroduce MluI or BamHI restriction sites for cloning into either Knobor full-length fiber constructs.

The exogenous peptides encode the adenovirus (Ad) capsid fiber and knobproteins. The fiber delineates each of the antenna-like projectionsextending from the Ad capsid that mediate primary cell binding toinitiate infection, and are comprised of an N-terminal long fibrousshaft domain followed by a C-terminal globular knob domain. The knobdomain is what specifically interacts with cell surface receptor and isproduced as a soluble protein in the absence of the shaft. Inv is aneffective ligand when fused to PBK10.

The Inv sequence is inserted into the knob using two strategies ofreplacing knob sub-domains with Inv, resulting in the recombinant knobconstructs, ABCJ-Inv and AGJ-Inv. Recombinant protein is produced inbacteria from pRSET vectors expressing these constructs, as bothN-terminally GFP-tagged (GFP-ABCJ-Inv and GFP-AGJ-Inv) and untaggedprotein. In a third strategy, Inv was inserted into the DG loop of theknob and used in vitro translation to produce S35-labeled protein.Native gel electrophoresis showed the recombinant protein maintained theformation of oligomers (dimers and trimers) similar to wild-type solubleknob (thus suggesting that the Inv insert does not confound the tertiarystructure of the fusion protein). In all scenarios, a full-lengthsoluble protein is produced.

Constructs in which Inv replaced the knob of the full-length fiberprotein were also created. A baculovirus vector was used to express theprotein in bacteria. A second fiber construct was created to express Invin between the shaft and knob domains of the full-length fiber protein,with a FactorX cleavage site introduced between Inv and Knob.

Example 6: Ligand for CD4

The amino acid sequence, TITLPCRIKQFINMWQEVGKAMYAPPISGQIRCSSNITGLLLTR(SEQ ID NO:5), derived from the CD4 binding domain of the humanimmunodeficiency virus (HIV) gp120 envelope protein, is sufficient forbinding to CD4. This sequence was obtained using standard molecularbiology techniques. The nucleic acid sequence encoding SEQ ID NO:5 (˜132nt) was cloned into pBluescript (PSK) plasmid. Ethidium bromide stainedelectrophoresis gel showed excision of insert (˜132 nt) from the vector(˜3 kb) by BamHI-EcoRI digest. The resulting product is then insertedin-frame with exogenous peptides such as PBK10 for targeting suchdelivery proteins to cells expressing CD4 (i.e. “helper” T-cells).

What is claimed is:
 1. A method of delivering a payload to the brain ofa subject having a brain disorder, comprising: systemicallyadministering a nanoparticle composition comprising a plurality ofnanoparticles comprising (a) a drug delivery polypeptide and (b) thepayload to the subject to allow the drug delivery polypeptide and thepayload to cross a blood-brain barrier in the subject, therebydelivering the payload to the brain of the subject, wherein the drugdelivery polypeptide comprises a ligand that targets HER3, a penton basesegment, and a payload binding domain.
 2. The method of claim 1, whereinthe payload comprises a chemotherapeutic agent.
 3. The method of claim2, wherein the chemotherapeutic agent is delivered to a cancer cell inthe brain.
 4. The method of claim 2, wherein the chemotherapeutic agentis delivered to a metastatic cancer cell in the brain.
 5. The method ofclaim 1, wherein the ligand is derived from heregulin alpha.
 6. Themethod of claim 1, wherein the payload comprises a chemotherapeuticagent intercalated into a double stranded DNA molecule.
 7. The method ofclaim 6, wherein the chemotherapeutic agent is doxorubicin.
 8. Themethod of claim 1, wherein the payload comprises a corrole.
 9. Themethod of claim 8, wherein the corrole comprises manganese.
 10. Themethod of claim 1, wherein the payload is delivered to the brain forimaging in the brain.
 11. The method of claim 1, further comprisingimaging the brain using magnetic resonance imaging.
 12. The method ofclaim 1, wherein the payload comprises a nucleic acid.
 13. The method ofclaim 12, wherein the payload comprises RNA.
 14. The method of claim 13,wherein the payload comprises siRNA.
 15. The method of claim 12, whereinthe payload comprises DNA.
 16. The method of claim 1, wherein the ligandcomprises an Ig-like domain and an EGF-like domain of heregulin-α. 17.The method of claim 1, wherein the payload binding domain comprises adecalysine motif.
 18. The method of claim 5, wherein the payload bindingdomain comprises a decalysine motif.
 19. The method of claim 1, whereinthe payload binds to the payload binding domain through electrostaticinteractions.
 20. The method of claim 1, wherein the penton base segmentis an adenovirus penton base protein or fragment thereof.
 21. The methodof claim 5, wherein the penton base segment is an adenovirus penton baseprotein or fragment thereof.
 22. The method of claim 17, wherein thepenton base segment is an adenovirus penton base protein or fragmentthereof.
 23. The method of claim 1, wherein the drug deliverypolypeptide is HerPBK10.
 24. The method of claim 1, wherein the braindisorder is a cancer in the brain.